System for verification of drug levels using dried blood samples

Abstract

Provided herein is a system including a mass spectrometry device configured to (i) generate a peak representing a drug in a DBS sample extracted from a pregnant subject after administration of a treatment for multiple sclerosis to the pregnant subject, and (ii) generate a peak representing an internal standard, a computer-readable memory containing computer-executable instructions, and one or more processors communicatively coupled to the mass spectrometry device and configured to execute the computer-executable instructions, wherein, when the one or more processors are executing the computer-executable instructions, the one or more processors are configured to perform operations including: determining a peak area ratio of the drug in the extracted DBS sample to the internal standard; and identifying the administered treatment as being below the internal standard threshold when the peak area ratio of the drug in the extracted DBS sample to the internal standard is less than 1. Also provided herein are dried blood spot cards and kits including dried blood spot cards pretreated with at least one internal standard.

Description

[Technical field]

[0001] Described herein are methods and kits for analyzing pharmaceutical molecules, such as teriflunomide, in dried blood samples. [Background technology]

[0002] Pregnancy brings many uncertainties to the pregnant subject and to the medical staff treating the pregnant subject for various pre-existing medical conditions (e.g., multiple sclerosis, diabetes, hypertension, cancer) and conditions that may arise during pregnancy (e.g., gestational diabetes, anemia, hyperemesis gravidarum). It may be beneficial for pregnant subjects and those planning a pregnancy to ensure that they are receiving the correct dose of medication and that their drug plasma concentrations remain within certain acceptable ranges in order to minimize and prevent harm to the unborn fetus and the pregnant subject. Summary of the Invention [Means for solving the problem]

[0003] The present disclosure relates, at least in part, to a method for the preparation of a compound comprising administering to a patient a predetermined amount of a stable label [ 13 C2, 2 The present invention is based on the discovery that plasma concentrations of teriflunomide in subjects (e.g., pregnant subjects) can be accurately and precisely monitored using a small drop of blood deposited on a filter paper pretreated with [H3]-teriflunomide (internal standard, ISTD). The pretreatment of the filter paper with ISTD is repeatable and reproducible. Dried blood spot (DBS) samples can be sent to any facility (e.g., laboratory) equipped with a high performance liquid chromatography mass spectrometer (LC-MS / MS) to determine teriflunomide levels without the need to use a validated method. The method described herein allows for rapid, reliable, reproducible, precise, non-invasive (e.g., finger prick) monitoring of plasma teriflunomide concentrations.

[0004] Spotting of blood onto pretreated filters was found to have no significant effect on the homogeneity of ISTD distribution or on the accuracy and precision of the method, and extreme hematocrit values ​​of the spotted blood (e.g., 25% and 65% of the hematocrit of whole blood) were found to have no significant effect on quantification of teriflunomide using pretreated filters.

[0005] Provided herein are methods of monitoring treatment of multiple sclerosis in a pregnant subject, the methods including (a) extracting a drug from a dried blood spot (DBS) sample, the DBS sample being from a pregnant subject after the pregnant subject has been treated for multiple sclerosis, (b) performing mass spectrometry on the extracted DBS sample, (c) determining a peak area ratio of the drug in the extracted DBS sample to an internal standard, and (d) identifying the administered treatment as below the internal standard threshold when the peak area ratio of the drug in the extracted DBS sample to the internal standard is less than 1.

[0006] Also provided herein are methods of determining the effectiveness of a treatment for multiple sclerosis in a pregnant subject, the methods including (a) extracting a drug from a dried blood sample, the dried blood sample being from a pregnant subject after the pregnant subject has been treated for multiple sclerosis, (b) performing mass spectrometry on the extracted DBS sample, (c) determining a peak area ratio of the drug in the extracted DBS sample to an internal standard, and (d) identifying the administered treatment as effective when the peak area ratio of the drug in the extracted DBS sample to the internal standard is less than 1.

[0007] Also provided herein are methods of analyzing blood samples from dried blood spots, the methods including (a) extracting a drug from a dried blood sample, the dried blood sample being from a pregnant subject after the pregnant subject has been treated for multiple sclerosis, (b) performing mass spectrometry on the extracted DBS sample, (c) determining a peak area ratio of the drug in the extracted DBS sample to an internal standard, and (d) identifying the administered treatment as below the internal standard threshold when the peak area ratio of the drug in the extracted DBS sample to the internal standard is less than 1.

[0008] In some embodiments of any of the methods described herein, the treatment administered is administration of a medication for relapsing-remitting multiple sclerosis.

[0009] In some embodiments of any of the methods described herein, the treatment or drug being administered is teriflunomide.

[0010] In some embodiments of any of the methods described herein, the methods further comprise, after (d), (e) administering to the pregnant subject an additional dose of teriflunomide.

[0011] In some embodiments, the additional dose of teriflunomide is from about 7 mg to about 14 mg of teriflunomide.

[0012] In some embodiments of any of the methods described herein, a peak area ratio of less than 1 indicates a level of teriflunomide in the pregnant subject that is harmless to a fetus of the pregnant subject.

[0013] In some embodiments of any of the methods described herein, the methods further include identifying the administered treatment as above the internal standard threshold when a peak area ratio of the drug in the extracted DBS sample to the internal standard is greater than 1.

[0014] In some embodiments of any of the methods described herein, the methods further comprise administering to the pregnant subject no further doses of teriflunomide.

[0015] In some embodiments, a peak area ratio greater than 1 indicates that the level of teriflunomide in the pregnant subject is harmful to a fetus of the pregnant subject.

[0016] In some embodiments of any of the methods described herein, the internal standard threshold is 0.02 mcg / mL of teriflunomide.

[0017] In some embodiments, teriflunomide is 2 H6]-teriflunomide or [ 13 C2, 2 H3]-teriflunomide. In some embodiments, teriflunomide is [ 2 H6]-teriflunomide. In some embodiments, teriflunomide is [ 13 C2, 2 H3]-teriflunomide.

[0018] Also provided herein are methods of monitoring treatment of any disease in a pregnant subject, the methods including: (a) extracting a drug from a dried blood spot (DBS) sample, the DBS sample being from a pregnant subject after treatment of the disease has been administered to the pregnant subject; (b) performing mass spectrometry on the extracted DBS sample; (c) determining a peak area ratio of the drug in the extracted DBS sample to an internal standard; and (d) identifying the administered treatment as being below the internal standard threshold when the peak area ratio of the drug in the extracted DBS sample to the internal standard is less than 1.

[0019] Also provided herein are methods for determining the effectiveness of a treatment of a disease in a pregnant subject, the methods including: (a) extracting a drug from a dried blood sample, the dried blood sample being from the pregnant subject after treatment of the disease has been administered; (b) performing mass spectrometry on the extracted DBS sample; (c) determining a peak area ratio of the drug in the extracted DBS sample to an internal standard; and (d) identifying the administered treatment as effective when the peak area ratio of the drug in the extracted DBS sample to the internal standard is less than 1.

[0020] Also provided herein are methods of analyzing blood samples from dried blood spots, the methods including: (a) extracting a drug from a dried blood sample, the dried blood sample being from a pregnant subject after treatment for a disease has been administered; (b) performing mass spectrometry on the extracted DBS sample; (c) determining a peak area ratio of the drug in the extracted DBS sample to an internal standard; and (d) identifying the administered treatment as below the internal standard threshold when the peak area ratio of the drug in the extracted DBS sample to the internal standard is less than 1.

[0021] In some embodiments of any of the methods described herein, the treatment administered is administration of a cardiac medication, an anticoagulant, a bronchodilator, an antibiotic, an antiepileptic drug, an antidepressant, an antimanic drug, an antipsychotic drug, an antiretroviral drug, or an immunomodulatory agent.

[0022] In some embodiments of any of the methods described herein, the methods further comprise, after (d), (e) administering to the pregnant subject an additional dose of the drug.

[0023] In some embodiments of any of the methods described herein, a peak area ratio of less than 1 indicates that the level of drug in the pregnant subject is harmless to a fetus of the pregnant subject.

[0024] In some embodiments of any of the methods described herein, the methods further include identifying the administered treatment as above the internal standard threshold when a peak area ratio of the drug in the extracted DBS sample to the internal standard is greater than 1.

[0025] In some embodiments of any of the methods described herein, the methods further comprise administering to the pregnant subject no further doses of the drug.

[0026] In some embodiments, a peak area ratio of greater than 1 indicates levels of drug in a pregnant subject that are harmful to a fetus of the pregnant subject.

[0027] Also provided herein are methods of monitoring the treatment of any disease in a subject, the methods including: (a) extracting a drug from a dried blood spot (DBS) sample, the DBS sample being from a subject after the subject has been treated for the disease; (b) performing mass spectrometry on the extracted DBS sample; (c) determining a peak area ratio of the drug in the extracted DBS sample to an internal standard; and (d) identifying the treatment as being below the internal standard threshold when the peak area ratio of the drug in the extracted DBS sample to the internal standard is less than 1.

[0028] Also provided herein are methods for determining the effectiveness of a treatment of a disease in a subject, the methods including: (a) extracting a drug from a dried blood sample, the dried blood sample being from the subject after treatment of the disease has been administered; (b) performing mass spectrometry on the extracted DBS sample; (c) determining a peak area ratio of the drug in the extracted DBS sample to an internal standard; and (d) identifying the administered treatment as effective when the peak area ratio of the drug in the extracted DBS sample to the internal standard is less than 1.

[0029] Also provided herein are methods of analyzing blood samples from dried blood spots, including (a) extracting a drug from a dried blood sample, the dried blood sample being from a subject after treatment for a disease has been administered, (b) performing mass spectrometry on the extracted DBS sample, (c) determining a peak area ratio of the drug in the extracted DBS sample to an internal standard, and (d) identifying the administered treatment as below the internal standard threshold when the peak area ratio of the drug in the extracted DBS sample to the internal standard is less than 1.

[0030] In some embodiments of any of the methods described herein, the treatment administered is administration of a cardiac medication, an anticoagulant, a bronchodilator, an antibiotic, an antiepileptic drug, an antidepressant, an antimanic drug, an antipsychotic drug, an antiretroviral drug, or an immunomodulatory agent.

[0031] In some embodiments of any of the methods described herein, the methods further comprise, after (d), (e) administering to the subject an additional dose of the drug.

[0032] In some embodiments of any of the methods described herein, a peak area ratio of less than 1 indicates a level of drug in the subject that is harmless to the subject.

[0033] In some embodiments of any of the methods described herein, the methods further include identifying the administered treatment as above the internal standard threshold when a peak area ratio of the drug in the extracted DBS sample to the internal standard is greater than 1.

[0034] In some embodiments of any of the methods described herein, the methods further comprise not administering to the subject an additional dose of the drug.

[0035] In some embodiments, a peak area ratio greater than 1 indicates that the level of drug in the subject is harmful to the subject.

[0036] In some embodiments of any of the methods described herein, the dried blood sample is a blood sample obtained from a finger prick.

[0037] In some embodiments of any of the methods described herein, the dried blood sample is a blood sample obtained from venipuncture.

[0038] In some embodiments of any of the methods described herein, the dried blood sample is a blood sample obtained from an arm stick, calf stick, thigh stick, or palm stick.

[0039] In some embodiments of any of the methods described herein, the dried blood sample is stored for a period of time prior to step (a).

[0040] In some embodiments, the period is from 12 hours to 14 days.

[0041] In some embodiments of any of the methods described herein, the dried blood sample is stored at room temperature.

[0042] Provided herein are methods for monitoring the treatment of multiple sclerosis in a pregnant subject. These methods include (a) extracting a drug from a dried blood spot (DBS) sample, where the DBS sample is from a pregnant subject after the pregnant subject has been treated for multiple sclerosis; (b) performing mass spectrometry on the extracted DBS sample; (c) determining a peak area ratio of the drug in the extracted DBS sample to a first internal standard; (d) determining a peak area ratio of the drug in the extracted DBS sample to a second internal standard; and (e) determining that the amount of drug in the pregnant subject is within an acceptable range when (i) the peak area ratio of the drug in the extracted DBS sample to the first internal standard is greater than 1 and (ii) the peak area ratio of the drug in the extracted DBS sample to the second internal standard is less than 1, where the peak area ratio of the drug in the extracted DBS sample to the first internal standard is associated with a minimum therapeutic efficacy level and the peak area ratio of the drug in the extracted DBS sample to the second internal standard is associated with a maximum therapeutic efficacy level.

[0043] Also provided herein are methods of determining the effectiveness of a treatment for multiple sclerosis in a pregnant subject, the methods including: (a) extracting a drug from a dried blood sample, the dried blood sample being from a pregnant subject after the pregnant subject has been treated for multiple sclerosis; (b) performing mass spectrometry on the extracted DBS sample; (c) determining a peak area ratio of the drug in the extracted DBS sample to a first internal standard; (d) determining a peak area ratio of the drug in the extracted DBS sample to a second internal standard; and (e) identifying the administered treatment as effective when (i) the peak area ratio of the drug in the extracted DBS sample to the first internal standard is greater than 1 and (ii) the peak area ratio of the drug in the extracted DBS sample to the second internal standard is less than 1, wherein the peak area ratio of the drug in the extracted DBS sample to the first internal standard is associated with a minimum therapeutic efficacy level and the peak area ratio of the drug in the extracted DBS sample to the second internal standard is associated with a maximum therapeutic efficacy level.

[0044] In some embodiments of any of the methods described herein, the treatment administered is administration of a medication for relapsing-remitting multiple sclerosis.

[0045] In some embodiments of any of the methods described herein, the administered treatment is identified as ineffective when the peak area ratio of the drug in the extracted DBS sample to the first internal standard and the peak area ratio of the drug in the extracted DBS sample to the second internal standard are less than 1, or when the peak area ratio of the drug in the extracted DBS sample to the second internal standard are greater than 1.

[0046] In some embodiments of any of the methods described herein, a peak area ratio of the drug in the extracted DBS sample relative to the first internal standard that is less than 1 indicates that the level of the drug in the pregnant subject is harmless to a fetus of the pregnant subject.

[0047] In some embodiments of any of the methods described herein, the methods further include identifying the administered treatment as above the second internal standard threshold when a peak area ratio of the drug in the extracted DBS sample to the second internal standard is greater than 1.

[0048] In some embodiments of any of the methods described herein, the methods further comprise administering to the pregnant subject no further doses of the drug.

[0049] In some embodiments of any of the methods described herein, a peak area ratio of the drug in the extracted DBS sample relative to the second internal standard that is greater than 1 indicates that the level of the drug in the pregnant subject is harmful to a fetus of the pregnant subject.

[0050] In some embodiments of any of the methods described herein, the first internal standard threshold is a minimum effective concentration of the drug.

[0051] In some embodiments of any of the methods described herein, the second internal standard threshold is a minimally toxic concentration of the drug.

[0052] In some embodiments of any of the methods described herein, the methods further comprise, after (e), (f) administering to the pregnant subject an additional dose of the drug.

[0053] Also provided herein are methods of monitoring treatment of a disease in a pregnant subject, the methods including: (a) extracting a drug from a dried blood spot (DBS) sample, the DBS sample being from the pregnant subject after treatment of the disease has been administered; (b) performing mass spectrometry on the extracted DBS sample; (c) determining a peak area ratio of the drug in the extracted DBS sample to a first internal standard; (d) determining a peak area ratio of the drug in the extracted DBS sample to a second internal standard; and (e) determining that the amount of drug in the pregnant subject is within an acceptable range when (i) the peak area ratio of the drug in the extracted DBS sample to the first internal standard is greater than 1 and (ii) the peak area ratio of the drug in the extracted DBS sample to the second internal standard is less than 1, wherein the peak area ratio of the drug in the extracted DBS sample to the first internal standard is associated with a minimum therapeutic efficacy level and the peak area ratio of the drug in the extracted DBS sample to the second internal standard is associated with a maximum therapeutic efficacy level.

[0054] Also provided herein are methods for determining the efficacy of a treatment of a disease in a pregnant subject, the methods including: (a) extracting a drug from a dried blood sample, the dried blood sample being from the pregnant subject after treatment of the disease has been administered; (b) performing mass spectrometry on the extracted DBS sample; (c) determining a peak area ratio of the drug in the extracted DBS sample to a first internal standard; (d) determining a peak area ratio of the drug in the extracted DBS sample to a second internal standard; and (e) identifying the administered treatment as effective when (i) the peak area ratio of the drug in the extracted DBS sample to the first internal standard is greater than 1 and (ii) the peak area ratio of the drug in the extracted DBS sample to the second internal standard is less than 1, the peak area ratio of the drug in the extracted DBS sample to the first internal standard being associated with a minimum therapeutic efficacy level and the peak area ratio of the drug in the extracted DBS sample to the second internal standard being associated with a maximum therapeutic efficacy level.

[0055] In some embodiments of any of the methods described herein, the treatment administered is administration of a cardiac medication, an anticoagulant, a bronchodilator, an antibiotic, an antiepileptic drug, an antidepressant, an antimanic drug, an antipsychotic drug, an antiretroviral drug, or an immunomodulatory agent.

[0056] In some embodiments of any of the methods described herein, the administered treatment is identified as ineffective when the peak area ratio of the drug in the extracted DBS sample to the first internal standard and the peak area ratio of the drug in the extracted DBS sample to the second internal standard are less than 1, or when the peak area ratio of the drug in the extracted DBS sample to the second internal standard are greater than 1.

[0057] In some embodiments of any of the methods described herein, a peak area ratio of the drug in the extracted DBS sample relative to the first internal standard that is less than 1 indicates that the level of the drug in the pregnant subject is harmless to a fetus of the pregnant subject.

[0058] In some embodiments of any of the methods described herein, the methods further include identifying the administered treatment as above the second internal standard threshold when a peak area ratio of the drug in the extracted DBS sample to the second internal standard is greater than 1.

[0059] In some embodiments of any of the methods described herein, the methods further comprise administering to the pregnant subject no further doses of the drug.

[0060] In some embodiments of any of the methods described herein, a peak area ratio of the drug in the extracted DBS sample relative to the second internal standard that is greater than 1 indicates that the level of the drug in the pregnant subject is harmful to a fetus of the pregnant subject.

[0061] In some embodiments of any of the methods described herein, the first internal standard threshold is a minimum effective concentration of the drug.

[0062] In some embodiments of any of the methods described herein, the second internal standard threshold is a minimally toxic concentration of the drug.

[0063] In some embodiments of any of the methods described herein, the methods further comprise, after (e), (f) administering to the pregnant subject an additional dose of the drug.

[0064] Also provided herein are methods of monitoring treatment of a disease in a subject, the methods including: (a) extracting a drug from a dried blood spot (DBS) sample, the DBS sample being from the subject after treatment of the disease has been administered; (b) performing mass spectrometry on the extracted DBS sample; (c) determining a peak area ratio of the drug in the extracted DBS sample to a first internal standard; (d) determining a peak area ratio of the drug in the extracted DBS sample to a second internal standard; and (e) determining that the amount of drug in the subject is within an acceptable range when (i) the peak area ratio of the drug in the extracted DBS sample to the first internal standard is greater than 1 and (ii) the peak area ratio of the drug in the extracted DBS sample to the second internal standard is less than 1, the peak area ratio of the drug in the extracted DBS sample to the first internal standard being associated with a minimum therapeutic efficacy level and the peak area ratio of the drug in the extracted DBS sample to the second internal standard being associated with a maximum therapeutic efficacy level.

[0065] Also provided herein are methods for determining the efficacy of a treatment of a disease in a subject, the methods including: (a) extracting a drug from a dried blood sample, the dried blood sample being from the subject after treatment of the disease has been administered; (b) performing mass spectrometry on the extracted DBS sample; (c) determining a peak area ratio of the drug in the extracted DBS sample to a first internal standard; (d) determining a peak area ratio of the drug in the extracted DBS sample to a second internal standard; and (e) identifying the administered treatment as effective when (i) the peak area ratio of the drug in the extracted DBS sample to the first internal standard is greater than 1 and (ii) the peak area ratio of the drug in the extracted DBS sample to the second internal standard is less than 1, the peak area ratio of the drug in the extracted DBS sample to the first internal standard being associated with a minimum therapeutic efficacy level and the peak area ratio of the drug in the extracted DBS sample to the second internal standard being associated with a maximum therapeutic efficacy level.

[0066] In some embodiments of any of the methods described herein, the treatment administered is administration of a cardiac medication, an anticoagulant, a bronchodilator, an antibiotic, an antiepileptic drug, an antidepressant, an antimanic drug, an antipsychotic drug, an antiretroviral drug, or an immunomodulatory agent.

[0067] In some embodiments of any of the methods described herein, the administered treatment is identified as ineffective when the peak area ratio of the drug in the extracted DBS sample to the first internal standard and the peak area ratio of the drug in the extracted DBS sample to the second internal standard are less than 1, or when the peak area ratio of the drug in the extracted DBS sample to the second internal standard are greater than 1.

[0068] In some embodiments of any of the methods described herein, a peak area ratio of the drug in the extracted DBS sample relative to the first internal standard that is less than 1 indicates that the level of the drug in the subject is harmless to the subject.

[0069] In some embodiments of any of the methods described herein, the methods further include identifying the administered treatment as above the second internal standard threshold when a peak area ratio of the drug in the extracted DBS sample to the second internal standard is greater than 1.

[0070] In some embodiments of any of the methods described herein, the methods further comprise not administering to the subject an additional dose of the drug.

[0071] In some embodiments of any of the methods described herein, a peak area ratio of the drug in the extracted DBS sample relative to the second internal standard that is greater than 1 indicates that the level of the drug in the subject is harmful to the subject.

[0072] In some embodiments of any of the methods described herein, the first internal standard threshold is a minimum effective concentration of the drug.

[0073] In some embodiments of any of the methods described herein, the second internal standard threshold is a minimally toxic concentration of the drug.

[0074] In some embodiments of any of the methods described herein, the methods further comprise, after (e), (f) administering to the subject an additional dose of the drug. In some embodiments of any of the methods described herein, the dried blood sample is a blood sample obtained from a finger prick.

[0075] In some embodiments of any of the methods described herein, the dried blood sample is a blood sample obtained from venipuncture.

[0076] In some embodiments of any of the methods described herein, the dried blood sample is a blood sample obtained from an arm stick, calf stick, thigh stick, or palm stick.

[0077] In some embodiments of any of the methods described herein, the dried blood sample has been stored for a period of time prior to step (a).

[0078] In some embodiments, the period is from 12 hours to 14 days.

[0079] Provided herein is a system including a mass spectrometry device configured to (i) generate a peak representative of a drug in a DBS sample extracted from a pregnant subject after a treatment for multiple sclerosis has been administered to the pregnant subject, and (ii) generate a peak representative of an internal standard; a computer readable memory including computer executable instructions; and one or more processors communicatively coupled to the mass spectrometry device and configured to execute the computer executable instructions, wherein when the one or more processors are executing the computer executable instructions, the one or more processors are configured to perform operations including: determining a peak area ratio of the drug in the extracted DBS sample to the internal standard; and identifying the administered treatment as being below the internal standard threshold when the peak area ratio of the drug in the extracted DBS sample to the internal standard is less than 1.

[0080] Provided herein is a system including a mass spectrometer configured to (i) generate a peak representative of a drug in a DBS sample extracted from a pregnant subject after a treatment for multiple sclerosis has been administered to the pregnant subject, and (ii) generate a peak representative of an internal standard; a computer readable memory including computer executable instructions; and one or more processors communicatively coupled to the mass spectrometer and configured to execute the computer executable instructions, wherein when the one or more processors are executing the computer executable instructions, the one or more processors are configured to perform operations including: determining a peak area ratio of the drug in the extracted DBS sample to the internal standard; and identifying the administered treatment as efficacious when the peak area ratio of the drug in the extracted DBS sample to the internal standard is less than 1.

[0081] In some embodiments of any of the systems described herein, the treatment or drug administered is teriflunomide.

[0082] In some embodiments, a peak area ratio of less than 1 indicates a level of teriflunomide in the pregnant subject that is harmless to the pregnant subject's fetus.

[0083] In some embodiments, a peak area ratio greater than 1 indicates that the level of teriflunomide in the pregnant subject is harmful to a fetus of the pregnant subject.

[0084] In some embodiments of any of the systems described herein, the internal standard is 0.02 mcg / mL of teriflunomide. 2 H6]-teriflunomide or [ 13 C2, 2 H3]-teriflunomide. In some embodiments, teriflunomide is [ 2 H6]-teriflunomide. In some embodiments, teriflunomide is [ 13 C2,2 H3]-teriflunomide.

[0085] Also provided herein is a system including a mass spectrometry device configured to (i) generate a peak representing a drug in a DBS sample extracted from a pregnant subject after a treatment for a disease is administered to the pregnant subject, and (ii) generate a peak representing an internal standard; a computer readable memory including computer executable instructions; and one or more processors communicatively coupled to the mass spectrometry device and configured to execute the computer executable instructions, wherein when the one or more processors are executing the computer executable instructions, the one or more processors are configured to perform operations including determining a peak area ratio of the drug in the extracted DBS sample to the internal standard, and identifying the administered treatment as being below the internal standard threshold when the peak area ratio of the drug in the extracted DBS sample to the internal standard is less than 1.

[0086] Provided herein is a system including a mass spectrometer configured to (i) generate a peak representing a drug in a DBS sample extracted from a pregnant subject after a treatment for a disease is administered to the pregnant subject, and (ii) generate a peak representing an internal standard; a computer readable memory including computer executable instructions; and one or more processors communicatively coupled to the mass spectrometer and configured to execute the computer executable instructions, wherein when the one or more processors are executing the computer executable instructions, the one or more processors are configured to perform operations including determining a peak area ratio of the drug in the extracted DBS sample to the internal standard, and identifying the administered treatment as effective when the peak area ratio of the drug in the extracted DBS sample to the internal standard is less than 1.

[0087] In some embodiments of any of the systems described herein, a peak area ratio of the drug in the extracted DBS sample relative to the first internal standard that is less than 1 indicates that the level of the drug in the pregnant subject is harmless to a fetus of the pregnant subject.

[0088] In some embodiments of any of the systems described herein, a peak area ratio of the drug in the extracted DBS sample relative to the second internal standard that is greater than 1 indicates that the level of the drug in the pregnant subject is harmful to a fetus of the pregnant subject.

[0089] In some embodiments of any of the systems described herein, the drug is a cardiac medication, an anticoagulant, a bronchodilator, an antibiotic, an antiepileptic drug, an antidepressant, an antimanic drug, an antipsychotic drug, an antiretroviral drug, or an immunomodulator.

[0090] Also provided herein is a system including a mass spectrometry device configured to (i) generate a peak representing a drug in a DBS sample extracted from a subject after a treatment for a disease has been administered, and (ii) generate a peak representing an internal standard; a computer readable memory including computer executable instructions; and one or more processors communicatively coupled to the mass spectrometry device and configured to execute the computer executable instructions, wherein when the one or more processors are executing the computer executable instructions, the one or more processors are configured to perform operations including determining a peak area ratio of the drug in the extracted DBS sample to the internal standard, and identifying the administered treatment as being below the internal standard threshold when the peak area ratio of the drug in the extracted DBS sample to the internal standard is less than 1.

[0091] Also provided herein is a system including a mass spectrometer configured to (i) generate a peak representing a drug in a DBS sample extracted from a subject after a treatment for a disease is administered, and (ii) generate a peak representing an internal standard; a computer readable memory including computer executable instructions; and one or more processors communicatively coupled to the mass spectrometer and configured to execute the computer executable instructions, wherein when the one or more processors are executing the computer executable instructions, the one or more processors are configured to perform operations including determining a peak area ratio of the drug in the extracted DBS sample to the internal standard, and identifying the administered treatment as effective when the peak area ratio of the drug in the extracted DBS sample to the internal standard is less than 1.

[0092] In some embodiments of any of the systems described herein, a peak area ratio of the drug in the extracted DBS sample relative to the first internal standard of less than 1 indicates that the level of the drug in the subject is harmless to the subject.

[0093] In some embodiments of any of the systems described herein, a peak area ratio of the drug in the extracted DBS sample relative to the second internal standard that is greater than 1 indicates that the level of the drug in the subject is harmful to the subject.

[0094] In some embodiments of any of the systems described herein, the drug is a cardiac medication, an anticoagulant, a bronchodilator, an antibiotic, an antiepileptic drug, an antidepressant, an antimanic drug, an antipsychotic drug, an antiretroviral drug, or an immunomodulator.

[0095] Provided herein is a mass spectrometer configured to (i) generate a peak representative of a drug in a DBS sample extracted from a pregnant subject after the pregnant subject has been treated for multiple sclerosis, (ii) generate a peak representative of a first internal standard, and (iii) generate a peak representative of a second internal standard; a computer readable memory including computer executable instructions; and one or more processors communicatively coupled to the mass spectrometer and configured to execute the computer executable instructions, wherein when the one or more processors are executing the computer executable instructions, the one or more processors perform the following functions: determine a peak area ratio of the drug in the extracted DBS sample relative to the first internal standard; A system is provided that is configured to perform operations including determining a peak area ratio of the drug in the extracted DBS sample to a second internal standard; and determining that the amount of the drug in the pregnant subject is within an acceptable range when (i) the peak area ratio of the drug in the extracted DBS sample to the first internal standard is greater than 1 and (ii) the peak area ratio of the drug in the extracted DBS sample to the second internal standard is less than 1, wherein the peak area ratio of the drug in the extracted DBS sample to the first internal standard is associated with a minimum therapeutic efficacy level and the peak area ratio of the drug in the extracted DBS sample to the second internal standard is associated with a maximum therapeutic efficacy level.

[0096] Also provided herein is a method and system for detecting multiple sclerosis comprising: (i) generating a peak representative of a drug in a DBS sample extracted from a pregnant subject after the pregnant subject has been treated for multiple sclerosis; (ii) generating a peak representative of a first internal standard; and (iii) generating a peak representative of a second internal standard; a computer readable memory including computer executable instructions; and one or more processors communicatively coupled to the mass spectrometer and configured to execute the computer executable instructions, wherein when the one or more processors are executing the computer executable instructions, the one or more processors calculate a peak area ratio of the drug in the extracted DBS sample relative to the first internal standard. determining a peak area ratio of the drug in the extracted DBS sample to the first internal standard; determining a peak area ratio of the drug in the extracted DBS sample to the second internal standard; and identifying the administered treatment as effective when (i) the peak area ratio of the drug in the extracted DBS sample to the first internal standard is greater than 1 and (ii) the peak area ratio of the drug in the extracted DBS sample to the second internal standard is less than 1, wherein the peak area ratio of the drug in the extracted DBS sample to the first internal standard is associated with a minimum therapeutic efficacy level and the peak area ratio of the drug in the extracted DBS sample to the second internal standard is associated with a maximum therapeutic efficacy level.

[0097] In some embodiments of any of the systems described herein, a peak area ratio of the drug in the extracted DBS sample relative to the first internal standard that is less than 1 indicates that the level of the drug in the pregnant subject is harmless to a fetus of the pregnant subject.

[0098] In some embodiments of any of the systems described herein, a peak area ratio of the drug in the extracted DBS sample relative to the second internal standard that is greater than 1 indicates that the level of the drug in the pregnant subject is harmful to a fetus of the pregnant subject.

[0099] In some embodiments of any of the systems described herein, the first internal standard is a minimum effective concentration of the drug.

[0100] In some embodiments of any of the systems described herein, the second internal standard is a minimally toxic concentration of the drug.

[0101] In some embodiments of any of the systems described herein, the treatment or drug administered is teriflunomide.

[0102] Also provided herein is a method for detecting a disease comprising: (i) generating a peak representing a drug in a DBS sample extracted from a pregnant subject after the pregnant subject has been treated for a disease; (ii) generating a peak representing a first internal standard; and (iii) generating a peak representing a second internal standard; a computer readable memory including computer executable instructions; and one or more processors communicatively coupled to the mass spectrometer and configured to execute the computer executable instructions, wherein when the one or more processors are executing the computer executable instructions, the one or more processors determine a peak area ratio of the drug in the extracted DBS sample relative to the first internal standard; A system is provided that is configured to perform operations including determining a peak area ratio of the drug in the extracted DBS sample to a second internal standard; and determining that the amount of the drug in the pregnant subject is within an acceptable range when (i) the peak area ratio of the drug in the extracted DBS sample to the first internal standard is greater than 1 and (ii) the peak area ratio of the drug in the extracted DBS sample to the second internal standard is less than 1, wherein the peak area ratio of the drug in the extracted DBS sample to the first internal standard is associated with a minimum therapeutic efficacy level and the peak area ratio of the drug in the extracted DBS sample to the second internal standard is associated with a maximum therapeutic efficacy level.

[0103] Also provided herein is a mass spectrometer configured to (i) generate a peak representative of a drug in a DBS sample extracted from a pregnant subject after the pregnant subject has been treated for a disease, (ii) generate a peak representative of a first internal standard, and (iii) generate a peak representative of a second internal standard; a computer readable memory including computer executable instructions; and one or more processors communicatively coupled to the mass spectrometer and configured to execute the computer executable instructions, wherein when the one or more processors are executing the computer executable instructions, the one or more processors determine a peak area ratio of the drug in the extracted DBS sample relative to the first internal standard. determining a peak area ratio of the drug in the extracted DBS sample to the first internal standard; determining a peak area ratio of the drug in the extracted DBS sample to the second internal standard; and identifying the administered treatment as effective when (i) the peak area ratio of the drug in the extracted DBS sample to the first internal standard is greater than 1 and (ii) the peak area ratio of the drug in the extracted DBS sample to the second internal standard is less than 1, wherein the peak area ratio of the drug in the extracted DBS sample to the first internal standard is associated with a minimum therapeutic efficacy level and the peak area ratio of the drug in the extracted DBS sample to the second internal standard is associated with a maximum therapeutic efficacy level.

[0104] In some embodiments of any of the systems described herein, a peak area ratio of the drug in the extracted DBS sample relative to the first internal standard that is less than 1 indicates that the level of the drug in the pregnant subject is harmless to a fetus of the pregnant subject.

[0105] In some embodiments of any of the systems described herein, a peak area ratio of the drug in the extracted DBS sample relative to the second internal standard that is greater than 1 indicates that the level of the drug in the pregnant subject is harmful to a fetus of the pregnant subject.

[0106] Also provided herein is a method and system for detecting a disease comprising: (i) generating a peak representative of a drug in a DBS sample extracted from a subject after the subject has been treated for a disease; (ii) generating a peak representative of a first internal standard; and (iii) generating a peak representative of a second internal standard; a computer readable memory including computer executable instructions; and one or more processors communicatively coupled to the mass spectrometer and configured to execute the computer executable instructions, wherein when the one or more processors are executing the computer executable instructions, the one or more processors determine a peak area ratio of the drug in the extracted DBS sample relative to the first internal standard; A system is provided that is configured to perform operations including determining a peak area ratio of a drug in an extracted DBS sample to two internal standards; and determining that the amount of drug in the subject is within an acceptable range when (i) the peak area ratio of the drug in the extracted DBS sample to a first internal standard is greater than 1 and (ii) the peak area ratio of the drug in the extracted DBS sample to a second internal standard is less than 1, wherein the peak area ratio of the drug in the extracted DBS sample to the first internal standard is associated with a minimum therapeutic efficacy level and the peak area ratio of the drug in the extracted DBS sample to the second internal standard is associated with a maximum therapeutic efficacy level.

[0107] Also provided herein is a method for detecting a peak area of ​​a drug in a DBS sample extracted from a subject after the subject has been treated for a disease, comprising: (i) generating a peak representing a drug in a DBS sample extracted from the subject after the subject has been treated for a disease; (ii) generating a peak representing a first internal standard; and (iii) generating a peak representing a second internal standard; a computer readable memory including computer executable instructions; and one or more processors communicatively coupled to the mass spectrometer and configured to execute the computer executable instructions, wherein when the one or more processors are executing the computer executable instructions, the one or more processors determine a peak area ratio of the drug in the extracted DBS sample relative to the first internal standard. determining a peak area ratio of the drug in the extracted DBS sample to the first internal standard; and identifying the administered treatment as effective when (i) the peak area ratio of the drug in the extracted DBS sample to the first internal standard is greater than 1 and (ii) the peak area ratio of the drug in the extracted DBS sample to the second internal standard is less than 1, wherein the peak area ratio of the drug in the extracted DBS sample to the first internal standard is associated with a minimum therapeutic efficacy level and the peak area ratio of the drug in the extracted DBS sample to the second internal standard is associated with a maximum therapeutic efficacy level.

[0108] In some embodiments of any of the systems described herein, a peak area ratio of the drug in the extracted DBS sample relative to the first internal standard of less than 1 indicates that the level of the drug in the subject is harmless to the subject.

[0109] In some embodiments of any of the systems described herein, a peak area ratio of the drug in the extracted DBS sample relative to the second internal standard that is greater than 1 indicates that the level of the drug in the subject is harmful to the subject.

[0110] In some embodiments of any of the systems described herein, the first internal standard is a minimum effective concentration of the drug.

[0111] In some embodiments of any of the systems described herein, the second internal standard is a minimally toxic concentration of the drug.

[0112] In some embodiments of any of the systems described herein, the treatment or drug administered is teriflunomide.

[0113] In some embodiments of any of the systems described herein, the drug is a cardiac medication, an anticoagulant, a bronchodilator, an antibiotic, an antiepileptic drug, an antidepressant, an antimanic drug, an antipsychotic drug, an antiretroviral drug, or an immunomodulator.

[0114] Provided herein is a dried blood spot (DBS) card comprising a filter paper and a pretreated area containing at least one internal standard of a drug, wherein the at least one internal standard of a drug is deposited in the pretreated area at a threshold level.

[0115] In some embodiments, the drug is a drug for the treatment of relapsing-remitting multiple sclerosis. In some embodiments of any of the DBS cards described herein, the drug is teriflunomide.

[0116] In some embodiments of any of the DBS cards described herein, the drug is a cardiac medication, an anticoagulant, a bronchodilator, an antibiotic, an antiepileptic drug, an antidepressant, an antimanic drug, an antipsychotic drug, an antiretroviral drug, or an immunomodulator.

[0117] In some embodiments of any of the DBS cards described herein, at least one internal standard threshold level is 0.02 mcg / mL of teriflunomide. 2 H6]-teriflunomide or [ 13C2, 2 H3]-teriflunomide. In some embodiments, teriflunomide is [ 2 H6]-teriflunomide. In some embodiments, teriflunomide is [ 13 C2, 2 H3]-teriflunomide. In some embodiments, the at least one internal standard comprises a first internal standard and a second internal standard.

[0118] In some embodiments, the first internal standard is the minimum effective concentration of the drug.

[0119] In some embodiments, the second internal standard is a minimally toxic concentration of the drug. In some embodiments of any of the DBS cards described herein, the DBS card comprises three or more pre-treated regions that contain at least one internal standard of a drug.

[0120] In some embodiments of any of the DBS cards described herein, the DBS card further includes patient identifying information.

[0121] Also provided herein is a kit comprising a dried blood spot card comprising a pretreated area containing at least one internal standard of a drug for a disease (e.g., multiple sclerosis) in a subject (e.g., a pregnant subject), wherein the at least one internal standard of the drug is deposited in the pretreated area at a threshold level associated with efficacy of the drug.

[0122] In some embodiments of any of the kits described herein, the medication for a disease is a cardiac medication, an anticoagulant, a bronchodilator, an antibiotic, an antiepileptic drug, an antidepressant, an antimanic drug, an antipsychotic drug, an antiretroviral drug, or an immunomodulator.

[0123] In some embodiments of any of the kits described herein, the disease is multiple sclerosis, hi some embodiments, the drug for multiple sclerosis is teriflunomide.

[0124] In some embodiments of any of the kits described herein, the subject is a pregnant subject.

[0125] In some embodiments, at least one internal standard is labeled with a radioisotope, a fluorophore, or a quencher.

[0126] In some embodiments of any of the kits described herein, the kit includes one or more of a single-use lancet, a syringe with a needle, a container, a desiccant pack, an alcohol swabs, a sterile gauze swabs, a patient form, technical instructions, and an envelope.

[0127] In some embodiments, the kit includes a single-use lancet, a container, a desiccant pack, an alcohol swab, a sterile gauze swab, a patient form, technical instructions, and an envelope.

[0128] In some embodiments of any of the kits described herein, the container is a gas-impermeable sealable bag. In some embodiments of any of the kits described herein, the container is a foil bag.

[0129] In some embodiments of any of the kits described herein, at least one internal standard is 0.02 mcg / mL teriflunomide.

[0130] In some embodiments of any of the kits described herein, the at least one internal standard is 2 H6]-teriflunomide or [ 13 C2, 2 H3]-teriflunomide.

[0131] In some embodiments of any of the kits described herein, the at least one internal standard comprises a first internal standard and a second internal standard.

[0132] In some embodiments, the first internal standard is the minimum effective concentration of the drug.

[0133] In some embodiments of any of the kits described herein, the second internal standard is a minimally toxic concentration of the drug.

[0134] As used herein, the word "a" before a noun refers to one or more of that particular noun. For example, the phrase "a small molecular" refers to "one or more small molecules."

[0135] The term "small molecule drug" as used herein refers to a therapeutic agent having a low molecular weight that is used in the prevention, diagnosis, or treatment of a pathological condition. Therapeutic agents are usually synthesized by organic chemistry, but may also be isolated from natural sources such as plants, fungi, and microorganisms.

[0136] The term "biologic drug" as used herein refers to any therapeutic substance made or obtained from a living organism or its products, used in the prevention, diagnosis, or treatment of a pathological condition. Thus, a biologic drug or preparation is a medical drug produced using biotechnology, e.g., proteins (e.g., recombinant therapeutic proteins) or nucleic acids (e.g., DNA, RNA, or antisense oligonucleotides), used for therapeutic or in vivo diagnostic purposes.

[0137] The terms "pregnant" or "pregnancy", as used herein, refer to the state of a subject having, exhibiting, or being determined to have a concentration of human placental lactogen, or related fragments thereof, human chorionic gonadotropin (hCG) hormone, or related fragments thereof, in a biological sample (e.g., urine, blood, bodily fluids) obtained from that subject in association with a period of pregnancy (e.g., 6 weeks or less, 6-9 weeks, 10-12 weeks, 13-16 weeks, 17-24 weeks).

[0138] The term "biological fluid" refers to any bodily fluid obtained from a mammalian subject (e.g., a human), such as blood, plasma, serum, or other blood fractions, urine, saliva, breast milk, or tears. In preferred embodiments, the biological fluid is urine, blood, serum, or plasma.

[0139] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this invention belongs.Methods and materials are described herein for use in this invention, but other suitable methods and materials known in the art can also be used.These materials, methods, and examples are merely illustrative and are not intended to be limiting.All published documents, patent applications, patents, sequences and database entries, and other references mentioned herein are incorporated herein by reference in their entirety.In case of discrepancy, the present specification, including definitions, will control.

[0140] Other aspects, features, and advantages of the invention will become apparent from the following detailed description and drawings, and from the claims. [Brief description of the drawings]

[0141] [Figure 1] FIG. 1 is a schematic representation of one example of a test system and process used to determine plasma teriflunomide concentrations in pregnant subjects. [Diagram 2] FIG. 1 is a schematic representation of one embodiment of a method for analyzing dried blood spot (DBS) samples using a high performance liquid chromatography mass spectrometer (LC-MS / MS) based on peak area ratios using a single internal standard (ISTD). [Diagram 3] FIG. 1 is a schematic representation of one embodiment of a method for analyzing DBS samples using LC-MS / MS based on peak area ratios using two ISTDs. [Figure 4]FIG. 1 is a schematic representation of a kit that can be used to determine plasma teriflunomide concentrations in pregnant subjects. [Diagram 5] FIG. 1 is a schematic representation of a system for pre-treating DBS cards with an ISTD. [Figure 6] FIG. 6 is a schematic representation showing the automated pipette head and microtip of the system of FIG. 5 for depositing ISTDs onto a DBS card. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0142] The present disclosure relates to systems and methods for monitoring drug plasma concentrations (e.g., plasma teriflunomide concentrations) using dried blood spots (DBS) in a subject (e.g., a pregnant subject or a subject planning a pregnancy).

[0143] Multiple sclerosis is a progressive and chronic demyelinating disease of the central nervous system that affects T cells (e.g., helper T cells) and B cells. Typically, multiple sclerosis is diagnosed in adolescents, e.g., people aged 20-50 years. Four stages of multiple sclerosis have been identified: clinically initial stage (CIS), relapsing remitting multiple sclerosis (RRMS), secondary progressive multiple sclerosis (SPMS), and primary progressive multiple sclerosis (PPMS). CIS is described as an episode of neurological symptoms lasting at least 24 hours. Here, subjects most often present with numbness or tingling in the arms, legs, or face, blurred vision, dizziness, and / or balance problems (Effendi, Noro Psikiyatr Ars., 2015, 52(Suppl 1): S1-S11). RRMS is defined by increasing neurological exacerbations followed by periods of partial or complete remission. During these neurological exacerbations, activated immune cells (e.g., T and B cells) cause localized areas of inflammation and damage that lead to the symptoms of multiple sclerosis. RRMS may last an average of 10 years, after which 50% of subjects with RRMS will develop permanent disability and develop SPMS (Lugaresi et al., Neuropsychiatr Dis Treat, 2013, 9: 893-914). 90% of subjects who experience RRMS will develop SPMS within 25 years. SPMS is characterized by a progressive deterioration of neurological function that may include intervals of relapse or remission. Approximately 15% of subjects diagnosed with multiple sclerosis have PPMS (Ontaned and Fox, Curr Opin Neurol., 2015, 28(3): 237-243).

[0144] There are various medications to treat exacerbations (i.e., inflammatory attacks associated with multiple sclerosis), including injectable, oral, and infusion medications. Non-limiting examples of injectable medications for multiple sclerosis include interferon beta-1a (e.g., AVONEX®, REBIF®, PLEGRIDY®), interferon beta-1b (e.g., BETASERON®, EXTAVIA®), and glatiramer acetate (e.g., COPAXONE®, GLATOPA®). Non-limiting examples of oral medications for multiple sclerosis include teriflunomide (e.g., AUBAGIO®), fingolimod (e.g., GILENYA®), cladribine (e.g., MAVENCLAD®), siponimod (e.g., MAYZENT®), dimethyl fumarate (e.g., TECFIDERA®, VUMERITY™). Non-limiting infusion medications include alemtuzumab (e.g., LEMTRADA®), mitoxantrone (e.g., NOVANTRONE®), ocrelizumab (e.g., OCREVUS®), and natalizumab (e.g., TYSABRI®). Other medications that can be used to treat multiple sclerosis include methylprednisolone (e.g., SOLU-MEDROL®), prednisone (e.g., DELTASONE®), and adrenocorticotropic hormone (ACTH) (e.g., HPACTHAR® gel). See, e.g., Bar-Or et al., Drugs 2014, 74(6): 659-674.

[0145] Teriflunomide (also known as (Z)-2-cyano-α,α,α-trifluoro-3-hydroxy-p-crotonotroidin) is an immunomodulatory drug that inhibits the mitochondrial enzyme, dihydroorotate dehydrogenase, required for pyrimidine de novo synthesis. Without directly causing cell death, teriflunomide slows the proliferation of activated T and B lymphocytes. Teriflunomide can be administered as an active agent or as leflunomide. Leflunomide is converted to teriflunomide in vivo upon administration via the opening of the isoxazole ring. Teriflunomide, sold under the name AUBAGIO®, was initially prescribed as a once-daily oral treatment for patients suffering from multiple sclerosis (e.g., relapsing-remitting multiple sclerosis). In some instances, teriflunomide is recommended at oral doses of 7 mg or 14 mg once daily. During pregnancy, it may be beneficial to maintain teriflunomide plasma concentrations below 0.02 mcg / mL.

[0146] Exemplary Systems and Processes Used to Determine Plasma Teriflunomide Concentrations in Pregnant Subjects 1 shows diagrammatically one example of a test system and process used to determine plasma teriflunomide concentrations in pregnant subjects 10. The process of determining plasma teriflunomide concentrations can be divided into several steps: (1) collection of blood onto a dried blood spot (DBS) card; (2) drying of the blood-stained DBS card; (3) storage and transport of the blood-stained DBS card; (4) extraction of teriflunomide and ISTD from the DBS card; and (5) analysis of the extracted teriflunomide sample.

[0147] As shown in FIG. 1, a pregnant subject 10 was first administered a stable labeled internal standard [ 13 C2, 2A subject deposits a droplet of his / her own blood on a DBS card 12 having a region 14 pretreated with [H3]-teriflunomide (ISTD). Any of a variety of techniques can be used to deposit the subject's blood on the DBS card 12, such as using a finger prick, palm prick, arm prick, calf prick, or thigh prick. Often a finger prick technique is used, in which the subject (or the subject's doctor, nurse, or other medical professional) pricks the subject's finger with a lancet device (e.g., a single-use auto-disabled lancet, a capillary blood sample collection device, or a reusable lancet). The subject 10 then positions the pricked finger over one of the pre-printed circles that indicate to the user the pre-treated region 14 on the DBS card 12, such that a droplet of blood drips onto the pre-treated region 14, creating a blood deposit 16 on the pre-printed circle that indicates the pre-treated region 14 on the DBS card 12. Due to the amount of ISTD used to form the pretreated area 14, the blood droplets tend to spread substantially across the entire surface of the pretreated area 14 of the DBS card 12. The blood droplets can drip onto each of the pre-circled pretreated areas 14 of the DBS card 12, creating multiple blood deposits 16. Although only three of the six pretreated areas 14 are shown with blood deposits 16 in FIG. 1, a user will typically deposit a blood droplet onto each of the pretreated areas 14 before sending the DBS card 12 for analysis. The DBS card 12 is left at room temperature for at least two hours to allow the blood deposits to dry. In many cases, the DBS card 12 is left at room temperature for three to four hours or overnight.

[0148] Once the blood deposit 16 has dried, the DBS card 12 with the blood deposited thereon is mailed or otherwise transmitted to a facility (e.g., a clinic, hospital, or research institute) having a laboratory that includes a liquid chromatography-mass spectrometry (LC-MS / MS) system 20 for analysis. Because the LC-MS / MS system 20 does not need to be highly specialized, in some cases the DBS processing center (e.g., a clinic, hospital, or research institute) may be located in the same building or office space where the blood was collected. Advantageously, as described below, any facility (e.g., a laboratory) equipped with a high performance liquid chromatography-mass spectrometer (e.g., a liquid chromatography tandem-mass spectrometer) can be used to determine plasma teriflunomide levels in the subject 10 without the need to use a validated method. While certain current plasma bioanalytical methods allow for precise determination of plasma teriflunomide concentrations, these blood samples must typically be sent to specialized vendors that are equipped to prepare plasma from blood samples and have access to validated methods and laboratories. The systems and methods described herein can provide results to subjects faster and more inexpensively than many of the current bioanalytical methods.

[0149] In some embodiments, processing of the blood-stained DBS card 12 is postponed until a later date. For example, the blood-stained DBS card 12 can be placed in a gas-tight sealable bag (e.g., a foil bag) that includes one or more desiccant pouches to protect the blood-stained DBS card 12 from moisture. Once the blood-stained DBS card 12 is placed in a gas-tight sealable bag (e.g., a foil bag), the bag containing the blood-stained DBS card can be stored at room temperature for up to four months.

[0150] Referring also to FIG. 2, which shows a more detailed schematic of the mass spectrometry system 20 and analysis, when the blood-stained DBS card 12 arrives at the laboratory, one or more portions 18 of the blood-stained pre-treated area 16 are punched out and then dropped into a container or tube 19 containing a solution (e.g., a solution containing methanol) to extract the teriflunomide and ISTD from the card. The diameter of the punched out portion 18 can range from 2 to 8 mm. In some embodiments, a 6 mm diameter portion is punched out from the center of the blood-stained pre-treated area 16. Any of a variety of punching techniques can be used. In some cases, one or more portions 18 of the blood-stained pre-treated area 16 are punched out from the center of the blood-stained pre-treated area 16 using a single-use 6 mm punching device. Examples of suitable punching devices that can be used include manual punching devices, such as the Harris Micro-Punch (Fisher Scientific), and semi-automatic punching devices, such as those available from TOMTEC, Inc.

[0151] To extract the drug (e.g., teriflunomide) and ISTD from the blood-stained DBS card 12, the punched-out blood-stained area of ​​the DBS card is extracted with a volume of methanol (e.g., 200 μL of methanol) contained within tube 19. The extracted sample can then be vortexed for about 20 minutes (e.g., 15, 16, 18, 20, 22, 24, or 25 minutes) and centrifuged at about 10,000 revolutions per minute (rpm) (e.g., about 12,000 rpm, 13,000 rpm, 14,000 rpm, or 15,000 rpm) for about 5 minutes. After centrifugation, an aliquot of the supernatant (i.e., the methanol containing teriflunomide, ISTD, and other soluble blood components in organic solvent) is injected into a high performance liquid chromatography mass spectrometer system 20.

[0152] With further reference to Figures 1 and 2, once the subject's DBS is extracted and collected from the DBS card 12, mass spectrometry is performed on the extracted DBS sample. Mass spectrometry is similarly performed on the ISTD. Chromatography can be performed on an Agilent Zorbax Eclipse XDB-C8 analytical column with 3.5 μm particle size (4.6 mm internal diameter, 50 mm length) at a flow rate of 0.7 mL / min with a mobile phase consisting of a mixture of water / acetonitrile (vol / vol) with 0.1% acetic acid. Optimal chromatographic separation can be achieved by running isocratically for 4.5 minutes. The column can be maintained at 40°C, with the column effluent being delivered to the mass spectrometer interface without bifurcation. Mass spectrometry conditions consist of a Sciex API4000 triple quadrupole mass spectrometer (MDS SCIEX; Applied Biosystems, Concord, ON, Canada) controlled by Analyst 1.4.2 software. The mass spectrometer can be operated in a negative multiple reaction monitoring mode by using a Turbo Ion Spray source. In some embodiments, the LC-MS / MS system 20 used to analyze the teriflunomide and ISTD samples includes a pump (e.g., Shimadzu LC-20AD pump), an autosampler (e.g., SIL-20AC autosampler), and an oven (e.g., CTO-20AC oven). The teriflunomide / ISTD area ratio is determined for each punched portion 18 of the blood-stained (16) pretreated region 14 of the DBS card 12. If the area ratio is less than 1, the teriflunomide concentration is considered to be below the cutoff (i.e., less than 0.02 mcg / mL). If the area ratio is greater than 1, the teriflunomide concentration is considered to be greater than the cutoff (i.e., greater than 0.02 mcg / mL). In the latter case, the concentration of teriflunomide can be estimated by multiplying the area ratio by the cutoff concentration (20 ng / mL). Based on the results of the mass spectrometry analysis, a decision can be made as to whether or not to increase / decrease the level of teriflunomide in the subject.For example, if the area ratio is less than 1, the teriflunomide concentration is considered to be below the cutoff (i.e., the teriflunomide concentration is less than 0.02 mcg / mL in the test sample), and therefore the level of teriflunomide in the subject can be increased and the subject can take an additional dose of teriflunomide.

[0153] If the area ratio is greater than 1, the teriflunomide concentration is considered to be greater than the cutoff (i.e., the teriflunomide concentration is greater than 0.02 mcg / mL in the test sample), and therefore the level of teriflunomide in the subject should be reduced. The subject should not take further doses of teriflunomide and / or should continue to monitor the level of teriflunomide after a period of time (e.g., after 2 weeks, 3 weeks, 4 weeks, 6 weeks, 8 weeks, or 10 weeks). In certain cases, an active step such as administering cholestyramine or activated charcoal to the subject can be performed to reduce the level of teriflunomide in the subject. In some cases, for example, 8 g of cholestyramine can be administered to the subject every 8 hours for 11 days. In other examples, 50 g of oral activated charcoal powder can be administered to the subject every 12 hours for 11 days.

[0154] FIG. 3 illustrates diagrammatically an alternative technique that can be performed by the LC-MS / MS system 20. This technique involves the use of a stable labeled internal standard [ 2 H6]-drug (ISTD) (IS-1) and stable labeled internal standard [ 13 C2, 2 The process includes spotting an H3]-drug (ISTD) (IS-2) onto each pretreated area 14 on the DBS card. IS-1 is a low internal standard concentration (e.g., a minimally effective ISTD concentration) and IS-2 is a high internal standard concentration (e.g., a minimally toxic or maximally therapeutic ISTD concentration). The process shown in FIG. 3 is essentially identical to the process shown in FIG. 2, except for the analysis of the extracted DBS samples. Therefore, only the analysis will be discussed in detail.

[0155] 3, if the area ratio of drug / IS-1 is less than 1, the drug concentration is considered to be less than the minimum effective concentration, and therefore the dose level of the drug in the subject should be increased. The subject should take an additional dose of the drug.

[0156] If the area ratio of drug / IS-2 is greater than 1, the drug concentration is considered to be greater than the minimum toxic concentration or greater than the maximum therapeutic concentration, and therefore the drug dosage level in the subject should be reduced. The subject should not take further doses of drug and / or should continue to monitor levels after a period of time defined by the subject's physician, nurse, or other medical professional (e.g., after 2 weeks, 3 weeks, 4 weeks, 6 weeks, 8 weeks, or 10 weeks).

[0157] If the area ratio of drug / IS-1 is greater than 1 and the area ratio of drug / IS-2 is less than 1, the drug concentration is considered to be within the therapeutic range or window, and thus the dose of the drug in the subject can be maintained or adjusted by the subject's physician, nurse, or other healthcare professional.

[0158] The determination of peak areas and peak area ratios, as well as the various other analyses described with reference to Figures 2 and 3, can be performed by a computer in the LC-MS / MS system 20. The computer can include one or more processors and one or more databases that enable the computer to perform the analyses. The computer can also be connected to a network that enables data and / or analysis results to be transmitted from the LC-MS / MS system 20 to other locations, such as a remote server.

[0159] 1 , the mass spectrometry results are configured in a report 30 that is sent to the pregnant subject 10 and / or a medical professional treating the pregnant subject 10. The report 30 may be a printed report that is mailed to the pregnant subject 10 or the pregnant subject's medical professional, or it may be an electronic file containing the data that is sent electronically to the pregnant subject 10 or the pregnant subject's medical professional. The information provided in the report can be used by the pregnant subject's medical professional to base future treatment on the level of teriflunomide determined to be in the subject's blood.

[0160] 4 illustrates a kit 100 that may be provided to a pregnant subject or a medical professional treating a pregnant subject to test the subject's plasma concentration (e.g., plasma teriflunomide concentration). The kit 100 includes a DBS card 12, a single-use lancet (e.g., a single-use finger prick tool) 102 that can be used to puncture the subject's skin, a syringe 104 having a needle that can be used for venipuncture, a container (e.g., a gas-impermeable sealable bag (e.g., a foil bag)) 106 having a desiccant pack 108, alcohol wipes 110, sterile gauze wipes 112, a patient form 114, technical instructions on how to collect a blood sample 116, and an envelope (e.g., a stamped envelope) 118 that can be used to mail the blood-stained DBS card to a laboratory for analysis. The various components of the kit 100 may be used to carry out the process described above for testing a pregnant subject's plasma teriflunomide concentration.

[0161] When a pregnant subject being treated for multiple sclerosis, for example, becomes pregnant or expresses an interest in becoming pregnant, the subject's physician can use the kit 100 to collect dried blood spot samples from the subject and then send these samples to a laboratory for testing. Typically, the physician will clean the subject's puncture site with an alcohol wipe 110 and then use the lancet 102 to collect a blood sample from the subject in the manner described above. However, in some cases, it may be desirable or necessary to collect blood from the subject by venipuncture by using a syringe 104. After collecting the blood spot sample, the dried DBS card 12 with the blood on it is placed in a bag 106 and the bag is sealed. The bag 106 containing the DBS card 12 with the blood on it is then placed in an envelope 118 along with a patient form 114 that has been completed to include the subject's identification, billing information, etc.

[0162] 5 shows a system 200 that can be used to generate a pretreated DBS card 12. Pretreated DBS cards can be made using various types of filter paper, such as Whatman 903 Protein saver filter paper and Perkin Elmer 226 filter paper. The filter paper is typically not impregnated with chemicals and does not have a wrap-around cover. Non-limiting types of filter paper that can be used include PerkinElmer 226 Bioanalysis RUO Card #226-1004 or PerkinElmer 226-5 Spot Ruo Card #226-1002.

[0163] 5, the system 200 includes an automated pipette head 202, a microtip 204 extending downward from the automated pipette head 202, a rack 206 for holding DBS cards, a DBS card elevator (e.g., protrusion) 208 for preventing contact between the DBS card 12 and the base of the rack, a container 210 for ISTD solution having a lid 216 with a through hole for receiving the microtip 204, and a preform that can be processed to form a DBS card 12. A card fixing edge 214 of the rack 206 serves to center the DBS card 12 within the rack 206. The automated pipette head 202 is equipped with variable tip spacing for use with various types of DBS cards as well as drip control and surface detection. The ISTD solution in the container 210 includes ISTD dissolved in methanol to a concentration of 80 ng / mL. In some examples, a highly concentrated stock solution of ISTD is first prepared in dimethyl sulfoxide, then 1 volume of this stock solution is diluted in 200 volumes of methanol solution, which can be used as the ISTD solution poured into the container 210. The container for the ISTD solution is equipped with a Peltier temperature control system 212 to maintain the temperature of the ISTD solution at 10°C to 15°C. The system 200 shown in Figure 5 is capable of depositing a precise volume of ISTD solution on the surface of the filter paper at the center of the deposition zone without significant splashing to form the pretreated area 14. Each microtip 204 is connected to an individual microsyringe, allowing the dispensing of precise micro-volumes of ISTD solution ranging from 5 μL to 25 μL.

[0164] Referring also to FIG. 6, to manufacture the DBS card 12, a preform DBS card formed from untreated filter paper is placed on a rack 206. The automated pipette head 202 is positioned over the ISTD container 210 and then lowered so that the microchips 204 are immersed in the ISTD solution. The microsyringes associated with the microchips 204 are then operated to draw a precise volume of ISTD solution into each of the microchips 204. The automated pipette head 202 is then raised to remove the microchips 204 from the container 210 and then moved laterally to position the microchips 204 over a designated area of ​​the preform DBS card. The microsyringes are then operated to dispense a desired volume of ISTD solution onto the filter paper to form the pretreated area 14.

[0165] If it is desired to pretreat the preform DBS card with multiple different ISTDs (e.g., to form a DBS card for use in the process described above with respect to FIG. 2), solutions of the different ISTDs (e.g., 13 C2, 2 H3]-teriflunomide and [ 2 A single container containing a mixture of ISTD solution (H4]-teriflunomide) can be provided and the automated pipette head 202 and microtip 204 can be manipulated to draw the ISTD solution into the microtip 204 and then deposit a precise volume of the ISTD solution onto the preformed DBS card.

[0166] The area on the DBS card 12 that will be pretreated with ISTDs 14 is typically dotted with ISTD solution at a defined concentration using the microtip 204 of the automated pipette head 202. The volume of ISTD solution dotted depends on the diameter of the pretreated area 14. For example, a pretreated area 14 with a circular diameter of 13 mm can be dotted with 20 μL of ISTDs. A pretreated area 14 with a circular diameter of 9 mm can be dotted with 15 μL of ISTDs. The diameter of the pretreated ISTD area 14 can be selected to be slightly larger than the diameter of the expected diameter of the blood deposit 16 to ensure that the correct and desired amount of blood and ISTDs is contained in the punched portion 18 of the DBS card 12. The process described above ensures that the ISTDs are homogeneously spread across the filter paper in the pretreated area 14 of the DBS card 12.

[0167] Methods for monitoring the treatment of multiple sclerosis Provided herein are methods for monitoring treatment of multiple sclerosis in a pregnant subject. In some examples, these methods include: (a) extracting a drug (e.g., teriflunomide) from a dried blood spot (DBS) sample, the dried blood sample being from a pregnant subject after the pregnant subject has been treated for multiple sclerosis; (b) performing mass spectrometry on the extracted DBS sample; (c) determining a peak area ratio of the drug (e.g., teriflunomide) in the extracted DBS sample to an internal standard; and (d) identifying the treatment as below the internal standard threshold when the peak area ratio of the drug in the extracted DBS sample to the internal standard is less than 1.

[0168] Also provided herein are methods for monitoring the treatment of multiple sclerosis in a pregnant subject. These methods include (a) extracting a drug from a dried blood spot (DBS) sample, the dried blood sample being from a pregnant subject after the pregnant subject has been treated for multiple sclerosis; (b) performing mass spectrometry on the extracted DBS sample; (c) determining a peak area ratio of the drug in the extracted DBS sample to a first internal standard; (d) determining a peak area ratio of the drug in the extracted DBS sample to a second internal standard; and (e) determining that the amount of drug in the pregnant subject is within an acceptable range when (i) the peak area ratio of the drug in the extracted DBS sample to the first internal standard is greater than 1 and (ii) the peak area ratio of the drug in the extracted DBS sample to the second internal standard is less than 1, wherein the peak area ratio of the drug in the extracted DBS sample to the first internal standard is associated with a minimum therapeutic efficacy level and the peak area ratio of the drug in the extracted DBS sample to the second internal standard is associated with a maximum therapeutic efficacy level.

[0169] Some embodiments further include recording (e.g., in a computer readable medium) the monitored multiple sclerosis status in the pregnant subject's medical record (e.g., the improved or stable multiple sclerosis status in the pregnant subject's medical record). Some examples further include notifying the subject, the subject's family, and / or the subject's primary care physician or attending physician of the pregnant subject's status after treatment is administered. Some embodiments further include authentication of refills of treatment administered. Some embodiments include discharging the pregnant subject from an inpatient facility (e.g., a hospital) based on the identity of the pregnant subject whose multiple sclerosis has improved or stabilized.

[0170] Method for determining the efficacy of a treatment for multiple sclerosis Provided herein are methods for determining the effectiveness of a treatment for multiple sclerosis in a pregnant subject. In some examples, these methods include: (a) extracting a drug (e.g., teriflunomide) from a dried blood spot (DBS) sample, the dried blood sample being from a pregnant subject after the pregnant subject has been treated for multiple sclerosis; (b) performing mass spectrometry on the extracted DBS sample; (c) determining a peak area ratio of the drug (e.g., teriflunomide) in the extracted DBS sample to an internal standard; and (d) identifying the administered treatment as effective when the peak area ratio of the drug in the extracted DBS sample to the internal standard is less than 1.

[0171] Also provided herein are methods of determining the efficacy of a treatment for multiple sclerosis in a pregnant subject, the methods including: (a) extracting a drug from a dried blood spot (DBS) sample, the dried blood sample being from a pregnant subject after the pregnant subject has been treated for multiple sclerosis; (b) performing mass spectrometry on the extracted DBS sample; (c) determining a peak area ratio of the drug in the extracted DBS sample to a first internal standard; (d) determining a peak area ratio of the drug in the extracted DBS sample to a second internal standard; and (e) identifying the administered treatment as effective when (i) the peak area ratio of the drug in the extracted DBS sample to the first internal standard is greater than 1 and (ii) the peak area ratio of the drug in the extracted DBS sample to the second internal standard is less than 1, wherein the peak area ratio of the drug in the extracted DBS sample to the first internal standard is associated with a minimum therapeutic efficacy level and the peak area ratio of the drug in the extracted DBS sample to the second internal standard is associated with a maximum therapeutic efficacy level.

[0172] Some embodiments of the methods described herein further comprise, after (e), (f) administering to the pregnant subject an additional dose of teriflunomide.

[0173] Some embodiments of any of these methods further include selecting a pregnant subject with multiple sclerosis or diagnosing the pregnant subject as having multiple sclerosis (e.g., using any of the methods for diagnosing multiple sclerosis known in the art). In some embodiments, the pregnant subject with multiple sclerosis may have previously been treated for multiple sclerosis without success.

[0174] Some embodiments further include recording the identified effectiveness of the administered treatment in the subject's medical record (e.g., a computer readable medium). Some examples further include notifying the subject, the subject's family, and / or the subject's primary care physician or attending physician of the identified effectiveness of the administered treatment. Some embodiments further include authorizing refills of the administered treatment identified as effective.

[0175] The period between treatment and testing can be, for example, 1-7 days, 1-5 days, 1-3 days, 1-2 days, 2-7 days, 2-4 days, 3-4 days, or 5-7 days.

[0176] Methods for monitoring the treatment of a disease Provided herein are methods for monitoring treatment of a disease in a subject (e.g., a pregnant subject). In some examples, these methods include: (a) extracting a drug (e.g., a cardiac drug, an anticoagulant, a bronchodilator, an antibiotic, an antiepileptic drug, an antidepressant, an antimanic drug, an antipsychotic drug, an antiretroviral drug, or an immunomodulator) from a dried blood spot (DBS) sample, the dried blood sample being from a subject (e.g., a pregnant subject) after treatment of the disease has been administered; (b) performing mass spectrometry on the extracted DBS sample; (c) determining a peak area ratio of the drug in the extracted DBS sample to an internal standard; and (d) identifying the administered treatment as being below the internal standard threshold when the peak area ratio of the drug in the extracted DBS sample to the internal standard is less than 1.

[0177] Also provided herein are methods of monitoring treatment of a disease in a subject (e.g., a pregnant subject). These methods include (a) extracting a drug (e.g., a cardiac drug, an anticoagulant, a bronchodilator, an antibiotic, an antiepileptic drug, an antidepressant, an antimanic drug, an antipsychotic drug, an antiretroviral drug, or an immunomodulator) from a dried blood spot (DBS) sample, the dried blood sample being from a subject (e.g., a pregnant subject) after treatment of the disease has been administered; (b) performing mass spectrometry on the extracted DBS sample; (c) determining a peak area ratio of the drug in the extracted DBS sample to a first internal standard; and (d) determining a peak area ratio of the drug in the extracted DBS sample to a second internal standard. and (e) determining that the amount of the drug in the subject (e.g., pregnant subject) is within an acceptable range when (i) a peak area ratio of the drug in the extracted DBS sample to the first internal standard is greater than 1 and (ii) a peak area ratio of the drug in the extracted DBS sample to the second internal standard is less than 1, wherein the peak area ratio of the drug in the extracted DBS sample to the first internal standard is associated with a minimum therapeutic efficacy level and the peak area ratio of the drug in the extracted DBS sample to the second internal standard is associated with a maximum therapeutic efficacy level.

[0178] Some embodiments further include recording (e.g., a computer readable medium) the monitored disease status in the subject's (e.g., pregnant subject's) medical record (e.g., improved or stable disease status in the subject's medical record). Some examples further include notifying the subject, the subject's family, and / or the subject's primary care physician or attending physician of the subject's status after treatment is administered. Some embodiments further include authentication of refills of treatment administered. Some embodiments include discharging the subject from an inpatient facility (e.g., a hospital) based on the identity of the subject with an improved or stable disease status.

[0179] Method for determining the efficacy of treatment of a disease Provided herein are methods for determining the effectiveness of a treatment in a subject (e.g., a pregnant subject). In some examples, these methods include: (a) extracting a drug (e.g., a cardiac drug, an anticoagulant, a bronchodilator, an antibiotic, an antiepileptic drug, an antidepressant, an antimanic drug, an antipsychotic drug, an antiretroviral drug, or an immunomodulator) from a dried blood spot (DBS) sample, the dried blood sample being from a subject (e.g., a pregnant subject) after treatment for a disease has been administered; (b) performing mass spectrometry on the extracted DBS sample; (c) determining a peak area ratio of the drug in the extracted DBS sample to an internal standard; and (d) identifying the administered treatment as effective when the peak area ratio of the drug in the extracted DBS sample to the internal standard is less than 1.

[0180] Also provided herein are methods for determining the effectiveness of a treatment of a disease in a subject (e.g., a pregnant subject). These methods include (a) extracting a drug (e.g., a cardiac drug, an anticoagulant, a bronchodilator, an antibiotic, an antiepileptic drug, an antidepressant, an antimanic drug, an antipsychotic drug, an antiretroviral drug, or an immunomodulator) from a dried blood spot (DBS) sample, the dried blood sample being from a subject (e.g., a pregnant subject) after treatment of the disease has been administered; (b) performing mass spectrometry on the extracted DBS sample; (c) determining the peak area ratio of the drug in the extracted DBS sample relative to a first internal standard; and (d) determining the peak area ratio of the extracted DBS sample relative to a second internal standard. and (e) identifying the administered treatment as effective when (i) a peak area ratio of the drug in the extracted DBS sample to the first internal standard is greater than 1 and (ii) a peak area ratio of the drug in the extracted DBS sample to the second internal standard is less than 1, wherein the peak area ratio of the drug in the extracted DBS sample to the first internal standard is associated with a minimum therapeutic effectiveness level and the peak area ratio of the drug in the extracted DBS sample to the second internal standard is associated with a maximum therapeutic effectiveness level.

[0181] Some embodiments of the methods described herein further comprise, after (e), (f) administering to the subject (e.g., a pregnant subject) an additional dose of the drug.

[0182] Some embodiments of any of these methods further include selecting a subject having the disease or diagnosing the subject as having the disease (e.g., using any of the methods for diagnosing disease known in the art). In some embodiments, a subject having the disease may have previously been treated for the disease, and the treatment was unsuccessful.

[0183] Some embodiments further include recording the identified effectiveness of the administered treatment in the subject's medical record (e.g., a computer readable medium). Some examples further include notifying the subject, the subject's family, and / or the subject's primary care physician or attending physician of the identified effectiveness of the administered treatment. Some embodiments further include authorizing refills of the administered treatment identified as effective.

[0184] The period between treatment and testing can be, for example, 1-7 days, 1-5 days, 1-3 days, 1-2 days, 2-7 days, 2-4 days, 3-4 days, or 5-7 days.

[0185] system Provided herein is a system including a mass spectrometry device configured to (i) generate a peak representing a drug (e.g., teriflunomide) in a dried blood spot (DBS) sample extracted from a pregnant subject after a treatment for multiple sclerosis has been administered to the pregnant subject, and (ii) generate a peak representing an internal standard; a computer readable memory including computer executable instructions; and one or more processors communicatively coupled to the mass spectrometry device and configured to execute the computer executable instructions, wherein when the one or more processors are executing the computer executable instructions, the one or more processors are configured to perform operations including determining a peak area ratio of the drug in the extracted DBS sample to the internal standard, and identifying the administered treatment as being below the internal standard threshold when the peak area ratio of the drug in the extracted DBS sample to the internal standard is less than 1.

[0186] Also provided herein is a system including a mass spectrometer configured to (i) generate a peak representing a drug (e.g., teriflunomide) in a dried blood spot (DBS) sample extracted from a pregnant subject after a treatment for multiple sclerosis has been administered to the pregnant subject, and (ii) generate a peak representing an internal standard; a computer readable memory including computer executable instructions; and one or more processors communicatively coupled to the mass spectrometer and configured to execute the computer executable instructions, wherein when the one or more processors are executing the computer executable instructions, the one or more processors are configured to perform operations including determining a peak area ratio of the drug in the extracted DBS sample to the internal standard and identifying the administered treatment as efficacious when the peak area ratio of the drug in the extracted DBS sample to the internal standard is less than 1.

[0187] Also provided herein is a method and system for detecting multiple sclerosis comprising: (i) generating a peak representative of a drug in a dried blood spot (DBS) sample extracted from a pregnant subject after the pregnant subject has been treated for multiple sclerosis; (ii) generating a peak representative of a first internal standard; and (iii) generating a peak representative of a second internal standard; a computer readable memory including computer executable instructions; and one or more processors communicatively coupled to the mass spectrometer and configured to execute the computer executable instructions, wherein when the one or more processors are executing the computer executable instructions, the one or more processors determine a peak area ratio of the drug in the extracted DBS sample relative to the first internal standard. determining a peak area ratio of the drug in the extracted DBS sample to the first internal standard; determining a peak area ratio of the drug in the extracted DBS sample to a second internal standard; and determining that the amount of the drug in the pregnant subject is within an acceptable range when (i) the peak area ratio of the drug in the extracted DBS sample to the first internal standard is greater than 1 and (ii) the peak area ratio of the drug in the extracted DBS sample to the second internal standard is less than 1, wherein the peak area ratio of the drug in the extracted DBS sample to the first internal standard is associated with a minimum therapeutic efficacy level and the peak area ratio of the drug in the extracted DBS sample to the second internal standard is associated with a maximum therapeutic efficacy level.

[0188] Also provided herein is a method and system for detecting multiple sclerosis comprising: (i) generating a peak representative of a drug in a dried blood spot (DBS) sample extracted from a pregnant subject after the pregnant subject has been treated for multiple sclerosis; (ii) generating a peak representative of a first internal standard; and (iii) generating a peak representative of a second internal standard; a computer readable memory including computer executable instructions; and one or more processors communicatively coupled to the mass spectrometer and configured to execute the computer executable instructions, wherein when the one or more processors are executing the computer executable instructions, the one or more processors generate a peak representative of the drug in the extracted DBS sample relative to the first internal standard. determining a peak area ratio of the drug in the extracted DBS sample to the first internal standard; determining a peak area ratio of the drug in the extracted DBS sample to the second internal standard; and identifying the administered treatment as effective when (i) the peak area ratio of the drug in the extracted DBS sample to the first internal standard is greater than 1 and (ii) the peak area ratio of the drug in the extracted DBS sample to the second internal standard is less than 1, wherein the peak area ratio of the drug in the extracted DBS sample to the first internal standard is associated with a minimum therapeutic efficacy level and the peak area ratio of the drug in the extracted DBS sample to the second internal standard is associated with a maximum therapeutic efficacy level.

[0189] Also provided herein is a system including a mass spectrometry device configured to (i) generate a peak representing a drug (e.g., a cardiac medication, an anticoagulant, a bronchodilator, an antibiotic, an antiepileptic, an antidepressant, an antimanic, an antipsychotic, an antiretroviral, or an immunomodulator) in a dried blood spot (DBS) sample extracted from a subject (e.g., a pregnant subject) after treatment for a disease has been administered, and (ii) generate a peak representing an internal standard; a computer readable memory including computer executable instructions; and one or more processors communicatively coupled to the mass spectrometry device and configured to execute the computer executable instructions, wherein when the one or more processors are executing the computer executable instructions, the one or more processors are configured to perform operations including determining a peak area ratio of the drug in the extracted DBS sample to the internal standard and identifying the administered treatment as being below the internal standard threshold when the peak area ratio of the drug in the extracted DBS sample to the internal standard is less than one.

[0190] Also provided herein is a system including: a mass spectrometer configured to (i) generate a peak representing a drug (e.g., a cardiac medication, an anticoagulant, a bronchodilator, an antibiotic, an antiepileptic, an antidepressant, an antimanic, an antipsychotic, an antiretroviral, or an immunomodulator) in a dried blood spot (DBS) sample extracted from a subject (e.g., a pregnant subject) after treatment for a disease has been administered, and (ii) generate a peak representing an internal standard; a computer readable memory including computer executable instructions; and one or more processors communicatively coupled to the mass spectrometer and configured to execute the computer executable instructions, wherein when the one or more processors are executing the computer executable instructions, the one or more processors are configured to perform operations including determining a peak area ratio of the drug in the extracted DBS sample to the internal standard and identifying the administered treatment as efficacious when the peak area ratio of the drug in the extracted DBS sample to the internal standard is less than one.

[0191] Also provided herein is a method for detecting a drug in a dried blood spot (DBS) sample from a subject (e.g., a pregnant subject) after treatment for a disease has been administered, comprising: (i) generating a peak representative of a drug (e.g., a cardiac drug, an anticoagulant, a bronchodilator, an antibiotic, an antiepileptic drug, an antidepressant, an antimanic drug, an antipsychotic drug, an antiretroviral drug, or an immunomodulatory drug); (ii) generating a peak representative of a first internal standard; and (iii) generating a peak representative of a second internal standard in a dried blood spot (DBS) sample from a subject (e.g., a pregnant subject) after treatment for a disease has been administered; a computer readable memory including computer executable instructions; and one or more processors communicatively coupled to the mass spectrometer and configured to execute the computer executable instructions, wherein when the one or more processors are executing the computer executable instructions, the one or more processors generate a peak representative of the first internal standard. determining a peak area ratio of the drug in the extracted DBS sample to the first internal standard; determining a peak area ratio of the drug in the extracted DBS sample to a second internal standard; and determining that the amount of the drug in the pregnant subject is within an acceptable range when (i) the peak area ratio of the drug in the extracted DBS sample to the first internal standard is greater than 1 and (ii) the peak area ratio of the drug in the extracted DBS sample to the second internal standard is less than 1, wherein the peak area ratio of the drug in the extracted DBS sample to the first internal standard is associated with a minimum therapeutic efficacy level and the peak area ratio of the drug in the extracted DBS sample to the second internal standard is associated with a maximum therapeutic efficacy level.

[0192] Also provided herein is a method for detecting a disease comprising: (i) generating a peak representing a drug (e.g., a cardiac medication, an anticoagulant, a bronchodilator, an antibiotic, an antiepileptic, an antidepressant, an antimanic medication, an antipsychotic medication, an antiretroviral medication, or an immunomodulator) in a dried blood spot (DBS) sample extracted from a subject (e.g., a pregnant subject) after treatment for a disease; (ii) generating a peak representing a first internal standard; and (iii) generating a peak representing a second internal standard; a computer readable memory comprising computer executable instructions; and one or more processors communicatively coupled to the mass spectrometer and configured to execute the computer executable instructions, wherein when the one or more processors are executing the computer executable instructions, the one or more processors: A system is provided that is configured to perform operations including determining a peak area ratio of a drug in an extracted DBS sample to a first internal standard; determining a peak area ratio of the drug in the extracted DBS sample to a second internal standard; and identifying a administered treatment as effective when (i) the peak area ratio of the drug in the extracted DBS sample to the first internal standard is greater than 1 and (ii) the peak area ratio of the drug in the extracted DBS sample to the second internal standard is less than 1, wherein the peak area ratio of the drug in the extracted DBS sample to the first internal standard is associated with a minimum therapeutic efficacy level and the peak area ratio of the drug in the extracted DBS sample to the second internal standard is associated with a maximum therapeutic efficacy level.

[0193] Dried Blood Spot (DBS) Cards The present invention relates to a method for preparing a filter paper and a drug (e.g., a stable label [ 13 C2, 2 H3]-teriflunomide (internal standard, ISTD) or [ 2 and a pre-deposited area containing at least one internal standard of a drug, such as teriflunomide (H6]-teriflunomide), wherein the at least one internal standard of a drug is deposited in the pre-deposited area at a threshold level.

[0194] In some instances, the drug is a drug for the treatment of relapsing-remitting multiple sclerosis (eg, teriflunomide).

[0195] In some examples of any of the DBS cards described herein, the at least one internal standard includes a first internal standard (e.g., a minimum effective concentration) and a second internal standard (e.g., a minimum toxic concentration or a maximum therapeutic concentration).

[0196] In some examples of any of the DBS cards described herein, the DBS card includes three or more pre-attached regions that contain at least one internal standard of a drug.

[0197] In some examples of any of the DBS cards described herein, the DBS card further includes patient identification information (eg, a barcode).

[0198] kit The development of a kit that allows direct comparison of the teriflunomide / ISTD ratio in unknown samples makes the analysis phase easy and suitable for any laboratory equipped with an LC-MS / MS system. Provided herein is a kit that essentially consists of or consists of at least a single DBS card (e.g., any of the DBS cards described herein) containing an internal standard (e.g., a single internal standard or at least two internal standards), a single-use lancet (e.g., a single-use finger prick tool), a syringe with a needle, a container (e.g., a gas-impermeable sealable bag (e.g., a foil bag)) with a desiccant pack, an alcohol swab, a sterile gauze swab, a patient form, technical instructions on how to collect blood samples, and an envelope (e.g., a stamped envelope). In some examples, the internal standard is labeled, for example, with a radioisotope, a fluorophore, or a quencher.

[0199] Other embodiments Although certain embodiments have been described, other embodiments are possible. For example, although some of the above processes have been described with respect to teriflunomide, it should be understood that the process can be used to detect the levels of other substances. For example, the process can be used to detect the levels of other drugs used to treat multiple sclerosis, such as fingolimod, cladribine, siponimod, and dimethyl fumarate. Similarly, the process can be used to detect the levels of other drugs that have long in vivo half-lives and may be problematic for pregnant subjects. Examples of such drugs include antiarrhythmics (e.g., digoxin), anticoagulants (e.g., warfarin), antiepileptics (e.g., carbamazepine, felbamate, lamotrigine, phenobarbital), antidepressants, antimanic or antipsychotics (e.g., citalopram, clomipramine, fluoxetine, lithium, nortriptyline, olanzapine, sertraline), antiretrovirals (e.g., nevirapine), immunomodulators (e.g., hydroxychloroquine, sirolimus, glucocorticoids).

[0200] The processes described herein can be used for any drug and / or any pharmaceutical agent for which it is beneficial to maintain the plasma concentration level of the drug and / or pharmaceutical agent within a certain concentration range and / or for which the therapeutic index is low. Examples of drugs with low therapeutic index include cardiac drugs (e.g., digoxin, disopyramide, procainamide), anticoagulants (e.g., warfarin, low molecular weight heparin, unfractionated heparin), bronchodilators (e.g., theophylline), antibiotics (e.g., amikacin, gentamicin, tobramycin, vancomycin, netilmicin), antiepileptics (e.g., carbamazepine, felbamate, gabapentin, lamotrigine, levetiracetam, oxcarbazepine, phenobarbital, phenytoin, valproic acid, and vigabatrin), antidepressants / antimanics / antipsychotics (e.g., Examples of suitable antiviral drugs include amitriptyline, bupropion, citalopram, clomipramine, clozapine, duloxetine, fluoxetine, haloperidol, imipramine, lithium, nortriptyline, olanzapine, paroxetine, quetiapine, risperidone, sertraline, venlafaxine), antiretrovirals (e.g., atazanavir, indinavir, nelfinavir, nevirapine, lopinavir, ritonavir, saquinavir), immunomodulators (e.g., azathioprine, cyclosporine, hydroxychloroquine, sirolimus, tacrolimus, corticosteroids). Similarly, it should be understood that it may be beneficial to maintain teriflunomide below certain threshold levels for reasons other than pregnancy.

[0201] Similarly, although the above process has been described according to collection of blood samples from a finger prick, other blood collection techniques can be used. For example, blood samples can be collected from a venous site, palm puncture, arm puncture, calf puncture, or femoral puncture. For subjects with distal limb irrigation failure, venipuncture can be an alternative performed by a skilled medical professional (e.g., a neurologist, physician, or nurse) and can be facilitated with a 1 mL syringe.

[0202] LC-MS / MS analysis can also be performed using an automated direct elution or online extraction system that serves as a front-end for the LC-MS / MS system. For one or the other method, a calibration of the MS / MS system is performed using standard solutions of teriflunomide and ISTD. Implementations of the subject matter and functional operations described herein can be implemented in digital electronic circuitry, in tangibly embodied computer software or firmware, in computer hardware including the structures disclosed herein and their structural equivalents, or in a combination of one or more of them. Software implementations of the subject matter described can be implemented as one or more computer programs. Each computer program can include one or more modules of computer program instructions encoded in a tangible, non-transitory computer-readable computer storage medium for execution by or for controlling the operation of a data processing device. Alternatively, or additionally, the program instructions can be encoded in / on an artificially generated propagating signal. For example, the signal can be a machine-generated electrical, optical, or electromagnetic signal generated to encode information for transmission to a receiver device suitable for execution by a data processing device. The computer storage medium can be a machine-readable storage device, a machine-readable storage substrate, a random or serial access memory device, or any combination of computer storage media.

[0203] The terms "data processing device", "computer", and "electronic computing device" (or equivalents as understood by those skilled in the art) refer to data processing hardware. For example, a data processing device can encompass any type of apparatus, device, and machine for processing data, including, by way of example, a programmable processor, computer, or multiple processors or computers. An apparatus can also include special purpose logic circuitry, including, for example, a central processing unit (CPU), a field programmable gate array (FPGA), or an application specific integrated circuit (ASIC). In some implementations, the data processing device or special purpose logic circuitry (or a combination of data processing devices or special purpose logic circuitry) can be hardware-based or software-based (or a combination of both hardware-based and software-based). The apparatus can optionally include code that creates an execution environment for a computer program, such as code that constitutes a processor firmware, a protocol stack, a database management system, an operating system, or a combination of the execution environment. The present disclosure contemplates the use of a data processing device with or without a conventional operating system, such as LINUX, UNIX, WINDOWS, MAC OS, ANDROID, or IOS.

[0204] A computer program, which may also be referred to or described as a program, software, software application, module, software module, script, or code, may be written in any form of programming language. Programming languages ​​may include, for example, compiled, interpreted, declarative, or procedural languages. A program may be arranged in any form, including as a stand-alone program, a module, a component, a subroutine, or a unit for use in a computing environment. A computer program may correspond to a file in a file system, but need not. A program may be stored in a portion of a file that holds other programs or data, for example one or more scripts stored in a markup language document, in a single file dedicated to the program, or in multiple cooperating files that store one or more modules, subprograms, or portions of code. A computer program may be deployed to run on one computer or on multiple computers, for example located at one site or distributed across multiple sites interconnected by a communications network. Although portions of the programs illustrated in the various figures may be illustrated as individual modules that implement various structures and functions through various objects, methods, or processes, the programs may instead include several sub-modules, third party services, components, and libraries. Conversely, structures and functions of various components may be combined into a single component as appropriate. The thresholds used to make the computational decision may be determined statically, dynamically, or both statically and dynamically.

[0205] The methods, processes, or logic flows described herein may be performed by one or more programmable computers executing one or more computer programs to perform functions by operating on input data and generating output. The methods, processes, or logic flows may also be performed by, and an apparatus may be implemented as, special purpose logic circuitry, such as a CPU, FPGA, or ASIC.

[0206] A computer suitable for executing a computer program can be based on one or more of general-purpose and special-purpose microprocessors and other types of CPUs. The elements of a computer are a CPU for performing or executing instructions and one or more memory devices for storing instructions and data. In general, a CPU can receive instructions and data from (and write data to) a memory. A computer can also include, or be operatively coupled to, one or more mass storage devices for storing data. In some implementations, a computer can receive data from and transfer data to a mass storage device, including, for example, a magnetic disk, a magneto-optical disk, or an optical disk. Furthermore, a computer can be incorporated in another device, for example, a mobile phone, a personal digital assistant (PDA), a mobile audio or video player, a game console, a global positioning system (GPS) receiver, or a portable storage device, such as a universal serial bus (USB) flash drive.

[0207] Computer readable media suitable for storing computer program instructions and data (transient or non-transient, as appropriate) can include all forms of permanent / non-permanent and volatile / non-volatile memory, media, and memory devices. Computer readable media can include semiconductor memory devices such as random access memory (RAM), read only memory (ROM), phase change memory (PRAM), static random access memory (SRAM), dynamic random access memory (DRAM), erasable programmable read only memory (EPROM), electrically erasable programmable read only memory (EEPROM), and flash memory devices. Computer readable media can also include magnetic devices such as tapes, cartridges, cassettes, and internal / removable disks. Computer readable media can also include magneto-optical disks, and optical memory devices, as well as technologies including digital video disks (DVD), CD ROM, DVD+ / -R, DVD-RAM, DVD-ROM, HD-DVD, and BLURAY. The memory may store a variety of objects or data, including caches, classes, frameworks, applications, modules, backup data, jobs, web pages, web page templates, data structures, database tables, repositories, and dynamic information. The types of objects and data stored in the memory may include parameters, variables, algorithms, instructions, rules, constraints, and references. Additionally, the memory may include logs, policies, security or access data, and report files. The processor and the memory may be supplemented by, or incorporated in, special purpose logic circuitry.

[0208] While the present invention has been described in conjunction with its detailed description, the above description is intended to be illustrative, and not limiting, of the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

Claims

1. 13. A system comprising: a mass spectrometry device configured to: (i) generate a peak representative of teriflunomide in a DBS sample extracted from a pregnant subject after the pregnant subject has been treated for multiple sclerosis; and (ii) generate a peak representative of an internal standard; A computer readable memory containing computer executable instructions; and one or more processors communicatively coupled to the mass spectrometry device and configured to execute computer-executable instructions; When the one or more processors are executing the computer-executable instructions, the one or more processors: Determining the peak area ratio of teriflunomide in the extracted DBS sample relative to an internal standard; identifying the administered treatment as being below the internal standard threshold when the peak area ratio of teriflunomide in the extracted DBS sample to the internal standard is less than 1; The system is configured to perform operations including:

2. 2. The system of claim 1, wherein a peak area ratio of less than 1 indicates that the level of teriflunomide in the pregnant subject is harmless to a fetus of the pregnant subject.

3. 2. The system of claim 1, wherein a peak area ratio greater than 1 indicates that a level of teriflunomide in the pregnant subject is harmful to a fetus of the pregnant subject.

4. The system according to any one of claims 1 to 3, wherein the internal standard is 0.02 µg / mL teriflunomide.

5. Teriflunomide is ２ H ６ ]-teriflunomide or [ １３ C ２ , ２ H ３ ]-teriflunomide.

6. Teriflunomide is ２ H ６ ]-teriflunomide.

7. Teriflunomide is １３ C ２ , ２ H ３ ]-teriflunomide.

8. 13. A system comprising: a mass spectrometer configured to: (i) generate a peak representative of a drug in a DBS sample extracted from a pregnant subject after the pregnant subject has been treated for multiple sclerosis; (ii) generate a peak representative of a first internal standard; and (iii) generate a peak representative of a second internal standard; A computer readable memory containing computer executable instructions; one or more processors communicatively coupled to the mass spectrometer and configured to execute computer-executable instructions; When the one or more processors are executing the computer-executable instructions, the one or more processors: determining a peak area ratio of the drug in the extracted DBS sample relative to a first internal standard; determining a peak area ratio of the drug in the extracted DBS sample relative to a second internal standard; (i) a peak area ratio of the drug in the extracted DBS sample to the first internal standard is greater than 1; (ii) the peak area ratio of the drug in the extracted DBS sample to the second internal standard is less than 1; identifying the administered treatment as effective, wherein a peak area ratio of the drug in the extracted DBS sample to the first internal standard is for a minimum therapeutic effectiveness level, and a peak area ratio of the drug in the extracted DBS sample to the second internal standard is for a maximum therapeutic effectiveness level; The system is configured to perform operations including:

9. 10. The system of claim 8, wherein a peak area ratio of the drug in the extracted DBS sample relative to the first internal standard of less than 1 indicates that the level of the drug in the pregnant subject is harmless to a fetus of the pregnant subject.

10. 10. The system of claim 8 or 9, wherein a peak area ratio of the drug in the extracted DBS sample relative to the second internal standard that is greater than 1 indicates that the level of the drug in the pregnant subject is harmful to a fetus of the pregnant subject.

11. The system according to any one of claims 8 to 10, wherein the first internal standard is a minimum effective concentration of the drug.

12. The system according to any one of claims 8 to 10, wherein the second internal standard is a minimally toxic concentration of the drug.

13. The system according to any one of claims 8 to 10, wherein the treatments and drugs administered are cardiac drugs, anticoagulants, bronchodilators, antibiotics, antiepileptic drugs, antidepressants, antimanic drugs, antipsychotic drugs, antiretroviral drugs, or immunomodulators.

14. The system according to any one of claims 8 to 10, wherein the treatment administered is administration of digoxin, warfarin, carbamazepine, felbamate, lamotrigine, phenobarbital, antidepressants, citalopram, clomipramine, fluoxetine, lithium, nortriptyline, olanzapine, sertraline, nevirapine, hydroxychloroquine, sirolimus, or glucocorticoids.

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