Quantitative determination of lysergide (LSD) and 2,3-dihydro-3-hydroxy-2-oxo lysergic acid diethylamide (O-H-LSD) in human plasma

Abstract

A method of measuring and identifying LSD and its primary metabolite O-H-LSD by obtaining a sample from an individual and measuring, identifying, and quantifying LSD and O-H-LSD in the sample by performing LC-MS / MS analysis. A method of treating and monitoring an individual taking LSD by administering a microdose of LSD, a prodrug of LSD, or an analog of LSD to the individual; monitoring the individual by obtaining a sample from an individual and measuring and identifying analytes in the sample by performing LC-MS / MS analysis; and adjusting the microdose based on the amount of LSD quantified in the LC-MS / MS analysis. A method of adjusting the dosing of LSD by administering a microdose of LSD, a prodrug of LSD, or an analog of LSD to the individual; and adjusting the microdose based on blood concentration analysis.

Description

[0001] Funding information

[0002] The research portion of this application was supported by a grant from the Swiss National Science Foundation (grant number 32003B_185111). Background of the Invention 1. Technical Field

[0004] This invention relates to compositions and methods for quantifying and identifying lysergic acid diethylamide (LSD) and its major metabolite 2,3-dihydro-3-hydroxy-2-oxoergot diethylamine (OH-LSD) in human plasma. 2. Background Technology

[0006] LSD is a typical hallucinogen (hallucinogen) widely used for recreational purposes (Krebs and Johansen, 2013). However, efforts are underway to use LSD and similar substances to treat depression and anxiety, substance use, and cluster headaches (Gasser et al., 2014; Liechti, 2017). Additionally, microdose administration of LSD has recently gained popularity for improving cognitive function and mood. In this regard, users take very low doses of 5-20 μg of LSD at 2- to 5-day intervals (Hutten et al., 2019). Furthermore, such microdose administration may also be used in the future for the treatment of medical conditions (Kuypers et al., 2019; Kuypers, 2020). For example, microdose LSD reduces pain perception (Ramaekers et al., 2021) and increases markers of human neurogenesis (Hutten et al., 2020).

[0007] With the rapid growth of interest in the application of LSD as a potential treatment for various mental disorders, expanding its clinical pharmacology, particularly its pharmacokinetic (PK) knowledge, is crucial. Therefore, measuring LSD exposure in patients and users is essential for investigating the association between drug exposure and therapeutic or toxic effects. PK data are needed to generate reference concentration values ​​to adjust the dosage for patients treated with LSD. For example, plasma concentrations can be measured in patients who do not show the expected strong psychoactive response or an inadequate treatment response to LSD. For this purpose, a method is needed to measure LSD at defined time points or repeatedly (C... maxThe concentration of LSD in plasma can be measured using a full pharmacokinetic (PPK) curve, and patient values ​​can then be compared with reference data from a larger population to determine the correct dosage and adjust the dosage of LSD adjunctive therapy within a therapeutic drug monitoring (TDM) framework. Furthermore, drug-drug interaction studies are still pending, which are crucial for ensuring safe and effective therapy. In this context, quantifying LSD metabolites (such as OH-LSD) is also important, as it helps interpret drug-drug interaction data. Finally, given that LSD (if available as a therapeutic agent) may be more readily available, appropriate bioanalytical methods are needed to identify drug abuse.

[0008] PK data have been established primarily for higher doses of LSD (Dolder et al., 2015; Dolder et al., 2017; Holze et al., 2019; Holze et al., 2021b). In contrast, PK data for microdose LSD are scarce (Family et al., 2020; Holze et al., 2021a). A key limitation in establishing PK data for microdose administration is the availability of a sensitive analytical method for detecting and effectively quantifying plasma LSD levels following administration of very low doses. This invention provides such a method.

[0009] Overall, the detection and reliable quantification of LSD are challenging, especially with microdose administration. Several studies have investigated the subjective and behavioral effects of microdose LSD (Bershad et al., 2019; Holze et al., 2021a; Yanakieva et al., 2019); however, only two studies have successfully reported plasma concentration-time curves for LSD (Family et al., 2020; Holze et al., 2021). In one study, plasma levels at a 5 μg LSD dose could not be determined due to insufficient sensitivity of the methods employed (Family et al., 2020), and for 10 and 20 μg treatments, only incomplete curves were established, partially covering the invasion and elimination of LSD. In another study, the quantification method was sensitive and comprised of the methods described in this paper, but only a subset of participants' plasma could be sampled (Holze et al., 2021). Therefore, more pharmacokinetic (PK) data on LSD, including microdose, are needed, along with a novel, sensitive, and well-functioning detection method.

[0010] Over the past few decades, several methods have been developed to quantify LSD and OH-LSD, such as Figure 1The above outlines the main points. Most methods focus on quantifying LSD for drug screening or preliminary pharmacokinetic studies involving limited sample volumes. In the 1990s, several gas chromatography-mass spectrometry (GC-MS / MS) methods were developed, primarily for quantifying LSD in urine (and plasma) and OH-LSD. These methods require large sample volumes of 2–10 ml and necessitate laborious extraction procedures involving either liquid-liquid or solid-phase extraction. The original extract must be evaporated and resuspended in a solvent suitable for GC analysis. Finally, in most cases, derivatization of the analyte is necessary to improve the resolution and sensitivity of the method. Around the millennium, the first GC-MS / MS and tandem mass spectrometry (LC-MS / MS) methods for LSD analysis in human fluids were developed. These methods require smaller sample volumes (approximately 1 ml) but still require complex extraction protocols involving either liquid-liquid or solid-phase purification of the biological sample. However, derivatization can be omitted compared to GC methods. Importantly, the total analysis time per sample is rarely less than 10 minutes. Over the past decade, novel LC-MS / MS methods have evolved, achieving lower limits of quantitation (LOQs) in the low pg / ml range. Notably, only a handful of methods have achieved LQs suitable for analyzing micro-dose LSD pharmacokinetics. As mentioned above, these methods utilize sophisticated sample preparation techniques and therefore still require a suitable sample volume (approximately 0.5 ml). In general, to our knowledge, no publicly available method is suitable for high-throughput analysis, and therefore these methods are unsuitable when analyzing large volumes of samples. Furthermore, due to their complex extraction procedures, these methods are impractical for routine therapeutic drug monitoring (TDM) analysis.

[0011] Therefore, novel and effective methods are still needed to assess LSD and OH-LSD in plasma, especially after treatment with microdose LSD. Summary of the Invention

[0012] The present invention provides a method for measuring and identifying LSD and its major metabolite OH-LSD by obtaining a sample from an individual and measuring, identifying and quantifying LSD and OH-LSD in the sample by performing LC-MS / MS analysis.

[0013] The present invention provides a method for treating and monitoring an individual taking LSD by administering a microdose of LSD, a prodrug of LSD, or an analogue of LSD to the individual; monitoring the individual by obtaining a sample from the individual and measuring and identifying the analyte in the sample by performing LC-MS / MS analysis; and adjusting the microdose based on the amount of LSD quantified in the LC-MS / MS analysis.

[0014] The present invention also provides a method for adjusting the dosage of LSD by administering a microdose of LSD, a prodrug of LSD, or an analogue of LSD to an individual; and adjusting the microdose based on blood concentration analysis. Attached Figure Description

[0015] Other advantages of the invention will be readily recognized and better understood when considered in conjunction with the accompanying drawings and with reference to the following detailed description, wherein:

[0016] Figure 1 This is a table comparing the present invention with previously disclosed analytical methods for quantitative analysis of LSD in human fluids or tissues;

[0017] Figure 2 It refers to LSD and OH-LSD in human plasma and their respective internal standards LSD-d3 and OH-LSD-d. 10 chromatogram of separation;

[0018] Figure 3 A-3B is a graph showing the calibration lines for LSD and OH-LSD in human plasma. Figure 3 A, on July 18, 2020; Figure 3 B, on July 20, 2020; and Figure 3 C, on July 21, 2020;

[0019] Figure 4 A-4D is a graph showing the selective determination of LSD and OH-LSD in human plasma treated with and without an internal standard (blank) (double blank), displaying seven OH-LSDs ( Figure 4 A) and LSD Figure 4 B) Overlay of double blank (thick black line) and LLOQ (dashed line) chromatograms, showing seven OH-LSD ( Figure 4 C) and LSD Figure 4 D) Overlay of blank (thick black line) and LLOQ (dashed line) chromatograms; and

[0020] Figure 5 This is a graph showing that the pharmacokinetics of three healthy volunteers who received an oral dose of 5 μg LSD can be established using the developed method. Detailed Implementation

[0021] This invention provides a method for measuring LSD and its metabolite OH-LSD in human samples (such as plasma). The method is validated, and information on its quality and performance, as well as its application in human subjects, is provided, including the first description of the pharmacokinetics of very low doses of LSD (including microdose of 5-25 μg LSD).

[0022] As used herein, “sample” means a sample of plasma, blood, urine, saliva or other bodily fluids from an individual, and preferably from a human or mammal.

[0023] As used herein, "metabolite" refers to an intermediate or final product of the original active compound that is the metabolite. The metabolite in this invention is preferably a metabolite of LSD, including OH-LSD. Furthermore, LSD and other prodrugs of LSD have been described or are under development. This method can also be used to determine the amounts of LSD and OH-LSD after administration of any other prodrug of LSD that produces the same metabolite or any other LSD analogue. Furthermore, the method can be modified to include the analysis of other ergotamine compounds. This includes analytical methods and the TDM concept for LSD-analog-assisted psychotherapy.

[0024] As used in this article, "LC-MS / MS" refers to liquid chromatography-tandem mass spectrometry analytical chemistry techniques.

[0025] This invention provides a method for measuring and identifying LSD and its metabolite OH-LSD by obtaining a sample from an individual and measuring and identifying the analytes in the sample by performing LC-MS / MS analysis. Compared to existing LC-MS / MS methods, this invention allows for sample processing in a less laborious manner, thus requiring less analysis time. Therefore, a well plate containing 96 samples can be processed in 40 minutes. This involves two steps: sample extraction (adding an extraction solvent to each sample) and plate centrifugation for 30 minutes. Furthermore, the sample analysis time (i.e., chromatographic run) is shorter than almost all existing methods, making this method suitable for high-throughput analysis. The analysis run time for each sample can be 4 minutes.

[0026] This invention requires a relatively small amount of sample material and remains more sensitive, or at least as sensitive, as other known methods. A sample volume of 300 μL from the subject is required, which is sufficient if reanalysis is necessary. 50 μL of sample can be used for practical LC-MS / MS methods. In terms of absolute sensitivity, this invention can quantify 0.5 pg LSD, while the limits of quantitation for existing methods are greater than 2.5 pg. This low limit of quantitation allows for the quantification of plasma levels of LSD after administration of a microdose that cannot be efficiently measured using existing methods. This high sensitivity also allows for the quantification of plasma levels of LSD longer after administration of any dose and expands the window for positive records of LSD use using human plasma. Quantification can be performed using this method up to six hours after administration. Importantly, methods using the same type of tandem mass spectrometer (API5500) did not reach our limit of quantitation, indicating that our extraction and chromatographic methods are more advantageous than others (Grumann et al., 2019) (Steuer et al., 2017). Finally, and importantly, this invention establishes reference PK data for future TDM (Therapeutic Drug Management). This analytical method and related TDM applications can be used to identify individuals who have taken LSD and whether their LSD levels are within the therapeutic range. Based on the amount of LSD quantified in this method, the dosage of LSD can be adjusted in individuals as needed. Furthermore, simultaneous determination of OH-LSD can be used to explain the impact of drug-drug interactions or diseases (such as hepatic or renal insufficiency) on the PK properties of LSD.

[0027] The LC-MS / MS method was thoroughly developed and fully validated in accordance with Regulatory Bioanalytical Guidance (FDA / EMA) for the analysis of LSD and OH-LSD in humans (EMA, 2011; FDA, 2018). This paper describes a state-of-the-art LC-MS / MS method for investigating the pharmacokinetic (PK) of LSD and OH-LSD. This method offers advantages over other prior art methods because it provides at least a 5-fold increase in sensitivity, uses small sample sizes, has a simple extraction protocol, and includes rapid sample analysis. To achieve these methodological advantages, plasma proteins are precipitated with acetonitrile. The sample is then centrifuged to solidify the precipitate at the bottom of the analysis tube, allowing the protein-free supernatant to be injected into the LC-MS / MS system. The injected sample is diluted online via a T-connector mounted in front of the analytical column, which enhances the interaction between the sample and the column. A pH-tolerant analytical column was selected to allow the use of a high pH of 9.0 (for mobile phase A). This further enhances the attraction and retention of LSD to the column, and thus also improves the sensitivity of the method. Overall, this invention enables a semi-automated workflow for extracting and analyzing samples in a 96-well plate format, facilitating high-throughput analysis. Relatedly, this method was put into practice and demonstrated in clinical studies by assessing the pharmacokinetic (PK) of microdose LSD in healthy participants. This demonstrates that a minimum dose of 5 μg of LSD in human plasma can be monitored effortlessly over long periods.

[0028] LSD is a prototype hallucinogenic drug that has been investigated as a treatment for a range of mental disorders (Gasser et al., 2014; Liechti, 2017). The pharmacokinetic properties of LSD, particularly at low doses, cannot be adequately characterized by just two preliminary studies (Family et al., 2020; Holze et al., 2021a). There is a need for efficient and rapid measurement of LSD plasma levels to analyze human plasma samples from pharmacokinetic studies and other clinical trials. OH-LSD is the major inactive metabolite of LSD, which is largely cleared by the kidneys.

[0029] Once LSD is marketed and regularly used in patients, TDM (Total Discharge Matrices) is needed to determine plasma concentrations. For example, plasma levels of the drug can be determined in patients who do not respond to regular doses of LSD, allowing for dose adjustment. However, a method is needed to reliably and rapidly measure LSD concentrations in plasma to provide such information to physicians. Therefore, the method must be simple for routine analysis. Furthermore, the metabolic ratio of LSD to OH-LSD can be used to identify slow or rapid metabolizers. The metabolic ratio also helps in dose adjustment in patients with renal or hepatic impairment. Finally, LSD and OH-LSD levels can be used to diagnose poisoning. Therefore, this invention was developed and validated, and includes a rapid LC-MS / MS method for quantifying LSD and OH-LSD in human plasma. The plasma sample is treated with acetonitrile via protein precipitation. The injected sample is then reacted with a pH-stable C... 18 An aqueous solution of ammonium bicarbonate (pH 9) was mixed in front of the analytical column to increase analyte retention. LSD and OH-LSD were detected by multiple reaction monitoring in positive and negative electrospray ionization modes, respectively.

[0030] This invention provides a method for treating and monitoring an individual taking LSD by administering a microdose of LSD, an LSD prodrug, or an LSD analogue to the individual; monitoring the individual by obtaining a sample from the individual and measuring and identifying the analyte in the sample by performing LC-MS / MS analysis; and adjusting the microdose based on the amount of LSD quantified in the LC-MS / MS analysis. This method can be used to slightly adjust the dosage and effect of LSD in an individual. Because microdose doses are very small, their efficacy or toxicity can vary significantly. Therefore, measuring the amount of LSD in the body and monitoring the individual to adjust the dosage is crucial.

[0031] The present invention also provides a method for adjusting the dosage of LSD by administering a microdose of LSD, a prodrug of LSD, or an analogue of LSD to an individual; and adjusting the microdose based on blood concentration analysis. As described above, blood concentration analysis is obtained by performing LC-MS / MS analysis.

[0032] As described in Example 1 below, inter-assay accuracy of 94.1%–104% and precision of ≤9.1% were recorded in three validation runs. Recovery was complete (≥98.3%), and importantly, recovery was consistent across different concentration levels and plasma batches (CV%: ≤3.84%). The plasma matrix caused minimal ion inhibition (-10.0%), and endogenous interferences were separable from the analyte. LSD and OH-LSD plasma samples could be thawed and refrozen for three cycles, held at room temperature for 8 hours, without showing degradation (≤8.83%). The linear range of this method (R ≥0.997) covers plasma concentrations observed in humans after microdose from 5 μg to high-dose LSD from 200 μg, and thus enables the assessment of the pharmacokinetics of LSD and OH-LSD. The LC-MS / MS method is convenient and reliable for measuring LSD and OH-LSD in plasma and contributes to the clinical development of LSD and TDM when used in patients.

[0033] The invention is further described in detail with reference to the following experimental examples. These examples are provided for illustrative purposes only and are not intended to be limiting unless otherwise stated. Therefore, the invention should not be construed as limited to the following examples, but should be interpreted as encompassing any and all variations that become apparent as a result of the teachings provided herein.

[0034] Example 1

[0035] Purpose

[0036] The aim of this study was to validate an analytical method for the simultaneous quantification of lysergic acid diethylamide (LSD) and 2,3-dihydro-3-hydroxy-2-oxoergot diethylamine (OH-LSD) in human plasma using an API 5500QTRAP LC-MS / MS system. This method is being used to analyze plasma samples from clinical studies using LSD. The analyses were performed at University Hospital Basel.

[0037] Overview of bioanalytical methods

[0038] A bioanalytical method for the simultaneous quantification of LSD and OH-LSD in human plasma samples by LC-MS / MS on an API 5500QTRAP tandem mass spectrometer was developed and validated. Calibration (Cal) and quality control (QC) samples were prepared in human plasma. Routine performance was controlled by analyzing the QC samples. 50 μl of human plasma was used to process the sample, while 50 μl aliquots were mixed with 150 μl of internal standard (ISTD) working solution. The samples were vortexed for approximately 1 minute and centrifuged to obtain a clear supernatant free of plasma proteins. 10 μl aliquots of the supernatant were injected into the LC-MS / MS system. All Cal and QC samples underwent the same assay procedure. The lower limit of quantitation (LLOQ) was set at 10 pg / mL, and the upper limit of quantitation (ULOQ) was set at 10,000 pg / mL. The analytical method was validated according to the standards outlined in the FDA Industry Guidance for Validation of Bioanalytical Methods (FDA, 2018), May 2018.

[0039] Reference Projects

[0040] The following reference materials were used to prepare the ISTN solution, as well as Cal and QC samples.

[0041] Table 1: Reference Materials

[0042]

[0043]

[0044] Blank human plasma

[0045] Obtain blank human plasma (anticoagulant: lithium heparin) from the local blood donation center (Blutspendezentrum SRK beider Basel, Hebelstrasse 10, 4056 Basel, Switzerland). Store the plasma at approximately -20°C.

[0046] Instruments, reagents and materials

[0047] LC-MS / MS system

[0048]

[0049] equipment

[0050]

[0051]

[0052] HPLC column

[0053]

[0054] chemicals

[0055]

[0056] Description of the LC-MS / MS system

[0057] Data collection method

[0058]

[0059] mobile phase

[0060]

[0061] Automatic sampler washing solution

[0062]

[0063] LC-MS / MS settings

[0064] Initial HPLC setup

[0065]

[0066] Table 2: HPLC pump gradient program and time events for LSD and OH-LSD analysis

[0067] time Module event parameter 0.00 MS valve switch A 0.50 pump Pump B 10 0.50 pump Pump C 0 0.50 pump Total flow 0.6 1.00 MS valve switch B 2.75 pump Pump B 95 3.00 MS valve switch A 3.50 pump Pump B 95 3.51 pump Pump B 10 4.00 controller Finish

[0068] Between 1.0 and 3.0 minutes of each run, introduce the HPLC stream into the mass spectrometer (right valve, position B), or otherwise into the solvent waste bottle.

[0069] Retention time of analytes

[0070]

[0071] Mass spectrometer settings

[0072]

[0073] Table 3 below lists the m / z values ​​of different ions used to monitor the concentrations of analytes and ISTD in human plasma. Chromatograms of LSD and OH-LSD are shown below. Figure 2 As shown.

[0074] Table 3: Analyte-specific settings for analyzing LSD and OH-LSD.

[0075]

[0076] Figure 2 This is a chromatogram of LSD (5000 pg / ml) and OH-LSD (5000 pg / ml) in human plasma. LSD-d3 and OH-LSD-d10 Used as internal standard. LSD and OH-LSD eluted after 1.78 min and 1.51 min, respectively. Chromatogram recorded on July 21, 2020.

[0077] Data acquisition and computation

[0078] Sample lists, collection methods, data collection, and quantification were generated using Ibrance's analytical software (version 1.7.1). The concentrations of LSD and OH-LSD in Cal and QC samples were calculated using the internal standard method. Mean, standard deviation, accuracy, and precision data for Cal and QC samples were calculated using Microsoft Excel Office 365 (Washington, USA).

[0079] Data Report

[0080] The analyte test results are rounded to three significant figures. Concentrations below 10 pg / ml are reported as "blq".

[0081] Preparation of stock solution and working solution

[0082] The concentrations of the solutions were based on the free and non-ionized forms of the drug. All solutions were prepared in 1.5 ml microtubes (Zalstadt, Nuremberg, Germany).

[0083] LSD stock solution

[0084] Stock solution for Cal samples: 0.1 mg / ml LSD in acetonitrile

[0085] The 0.1 mg / ml LSD solution in acetonitrile was purchased from Lipomed (Allersheim, Switzerland).

[0086] QC sample stock solution: 1 mg / ml LSD in acetonitrile

[0087] 1.0 mg LSD by precise weight was purchased from Lipomed (Allersheim, Switzerland) and dissolved in 985 μl of acetonitrile (LSD purity: 98.5%).

[0088] ISTD stock solution: 0.1 mg / ml LSD-d3 in acetonitrile

[0089] The 0.1 mg / ml LSD-d3 solution in acetonitrile was purchased from Lipomed (Allersheim, Switzerland).

[0090] OH-LSD stock solution

[0091] Stock solution of Cal sample: 1 mg / ml OH-LSD in DMSO

[0092] 1.094 mg of OH-LSD by exact weight was purchased from Toronto Research Chemicals, Inc. (Ontario, Canada) and dissolved in 1050 μl of DMSO (OH-LSD purity: 96%).

[0093] QC sample stock solution: 1 mg / ml OH-LSD in DMSO

[0094] 1.233 mg of OH-LSD by exact weight was purchased from Toronto Research Chemicals, Inc. (Ontario, Canada) and dissolved in 1184 μl of DMSO (OH-LSD purity: 96%).

[0095] ISTD stock solution: 1 mg / ml OH-LSD-d in –MSO 10

[0096] 1 mg of OH-LSD-d 10 Dissolve in 1000 μl DMSO.

[0097] Shake the above preparation until it is completely dissolved, and then store it in a refrigerator at -20°C.

[0098] working solution

[0099] Stock solution mixture of Cal samples (Mix-C): 2500 ng / ml LSD and OH-LSD

[0100] The LSD (0.1 mg / ml) and OH-LSD (1 mg / ml) stock solutions were separately diluted in DMSO to a final concentration of 10 μg / ml. Therefore, 50 μl of LSD (0.1 mg / ml) was mixed with 450 μL of DMSO, and 10 μl of OH-LSD (1 mg / ml) was added to 990 μl of DMSO. Subsequently, 250 μL of each working solution (10 μg / ml) was mixed with 500 μL of DMSO. The resulting solution had a concentration of 2500 ng / ml for both LSD and OH-LSD.

[0101] Stock solution mixture for QC samples (Mix-Q): 2500 ng / ml LSD and OH-LSD.

[0102] LSD and OH-LSD stock solutions (1 mg / ml) were individually diluted in DMSO to a final concentration of 10 μg / ml. Therefore, 10 μl of each stock solution was added to 990 μL of DMSO. Subsequently, 250 μL of each working solution (10 μg / ml) was mixed with 500 μL of DMSO. The resulting solution had a concentration of 2500 ng / ml for both LSD and OH-LSD.

[0103] Shake the above preparation until it is completely dissolved, and then store it in a refrigerator at -20°C.

[0104] Preparation of calibration samples

[0105] Cal samples with concentrations ranging from 10 to 10,000 pg / ml were prepared using Mix-C working solution. Dilution procedures are reported in Tables 4A and 4B.

[0106] Tables 4A and 4B: Preparation of Cal Samples

[0107]

[0108]

[0109] The working solution was stored in 1.5 ml microtubes (Zalstein, Germany) at approximately -20 °C (Table 4A). The volume reported in Table 4B was used to prepare 2 ml Cal samples in human plasma. 50 μl aliquots were stored in 0.75 ml microtubes at approximately -20 °C.

[0110] Preparation of quality control samples

[0111] QC samples with five different concentrations of LSD and OH-LSD were prepared using Mix-Q working solutions. Working solutions were prepared as described in Table 5A, while QC samples in plasma were prepared according to Table 5B.

[0112] Tables 5A and 5B: Preparation of QC Samples

[0113]

[0114] The working solution was stored at approximately -20°C in 1.5 ml microtubes (Zalstein, Germany) (Table 5A). The volume reported in Table 5B was used to prepare 4 ml QC samples in human plasma. 50 μl aliquots were stored at approximately -20°C in 0.75 ml Thermo microtubes.

[0115] Preparation of internal standard solution

[0116] ISTD working solutions: 100 pg / ml LSD-d3 and 250 pg / ml OH-LSD-d3 in acetonitrile 10

[0117] Prepare 50 μl of LSD-d3 stock solution (0.1 mg / mL) in 450 μl of acetonitrile to obtain a working solution of 10 μg / mL. Prepare 10 μl of OH-LSD-d3 in 990 μl of acetonitrile. 10 Prepare a stock solution (1 mg / ml) to obtain a 10 μg / ml solution.

[0118] Mix 5 μL of LSD-d3 working solution (10 μg / ml) and 12.5 μL of OH-LSD-d 10 The working solution (10 μg / ml) was added to 500 ml of acetonitrile to obtain solutions of 100 pg / ml and 250 pg / ml, respectively. The solutions were stored at approximately -20°C.

[0119] Sample extraction

[0120] Thaw the plasma samples used for validation runs and process them as described in 1-4 below.

[0121] 1. Thaw the individual Cal and QC samples (50 μl aliquots).

[0122] 2. Add 150 μl ISTD (blank: acetonitrile).

[0123] 3. The vortex should last at least 30 seconds.

[0124] 4. Centrifuge at 10℃ and 3220g for 30 minutes.

[0125] If not used immediately, the treated sample should be stored at approximately 10°C.

[0126] Principles and calculations

[0127] Components of analysis run and verification run

[0128] The analytical run included two sets of 10 Cal samples, two double-blank samples (without ISTD), two blank samples (with ISTD), and at least three QC samples at three different concentrations (low, medium, and high). For the validation run, samples at five concentration levels (LLOQ, QC) were investigated. 低 QC 中 QC 高 Seven QC samples (ULOQ) were prepared. The QC samples were placed between the two sets of Cal samples. Blank samples were run before and after calibration. Cal and QC samples were processed and analyzed in the same manner.

[0129] Acceptance criteria for verification operation

[0130] The following conditions must be met:

[0131] The percentage deviation of the lowest calibration point from the nominal value must be within ±20%.

[0132] Other Cal samples must deviate from the nominal value by a percentage within ±15%.

[0133] At least 75% of all Cal samples (including the highest and lowest) must meet the above criteria.

[0134] The correlation coefficient (R) of the Cal curve must be greater than 0.99.

[0135] ≥67% of a QC sample at a concentration level (e.g., 5 out of 7) must be within ±15% of its theoretical value. For LLOQ, the concentration must be within 20%.

[0136] The analyte signal intensity in a double blank sample must be less than 20% of the quantitation limit.

[0137] Analysis and operation acceptance criteria

[0138] ≥67% of all QC samples (e.g., 5 out of 7) must be within ±15% of the theoretical value. 33% of QC samples (not all replicates at the same concentration) may exceed ±15% of the theoretical value and will otherwise be re-injected or a complete re-analytical run will be performed.

[0139] Calculation of calibration samples

[0140] Linear regression was performed using MultiQuant software (version 3.0.3) by plotting the measured peak area ratio of each analyte and its respective deuterated ISTD relative to the nominal concentration. LSD-d3 was used to normalize the LSD reaction, while OH-LSD-d 10 Used for OH-LSD normalization. Choose a weighting factor of 1 / x. 2 For linear regression. All compliant Cal samples are used to generate the standard calibration curve. This means that for a valid run, the standard calibration curve consists of at least 15 and at most 20 Cal samples. Cal samples outside the specification are not used for any further calculations.

[0141] Calculation of quality control samples

[0142] By using the appropriate peak area ratio, the calibration curve equation is used to invert and calculate the concentrations of LSD and OH-LSD in the QC samples. The obtained values ​​for each QC sample are then checked against the acceptance criteria.

[0143] Study performance calculation

[0144] accuracy

[0145] Precision is defined as reproducibility within and between assays. Calculate the mean, standard deviation, and percentage relative standard deviation (%CV) for each QC concentration (within assay) and three validation runs (between assays).

[0146] Accuracy

[0147] Accuracy is calculated by dividing the total average value of each QC level by the nominal value in each measurement (within measurement) and three validation runs (between measurement).

[0148] Selective I

[0149] In at least six different samples of drug-free human plasma, there should be no interference greater than 20% of the analyte peak area at the LLOQ level.

[0150] Selective II

[0151] The average accuracy of at least six different samples at the LLOQ level should be between 80% and 120%. ≥67% of these samples (e.g., 5 out of 7) must have an accuracy between 80% and 120%.

[0152] Residue

[0153] Residue between samples is determined by injecting a ULOQ sample, followed by two double-blank samples. The signal intensity of the analyte peak in the double-blank samples is compared to the signal intensity measured at the ULOQ level. The total residue of the analytical system used is typically approximately 0.1%. Additionally, the peak area of ​​the analyte in the double-blank samples is compared to the peak area measured at the LLOQ level. The residue should be less than 20% of the LLOQ peak area; otherwise, an additional solvent sample must be included for analysis of the studied samples.

[0154] Recovery rate and matrix effect

[0155] According to the guidelines used (FDA, 2018), the recoveries of the analyte and internal standard should be consistent, accurate and reproducible.

[0156] The matrix effect should be consistent across at least six batches of matrix. The %CV of the matrix effect calculated based on at least six batches of matrix should not exceed 15%. This determination should be performed at at least low and high concentration levels (EMA, 2011).

[0157] Stability test

[0158] Each analyte must be stable in human plasma for at least three freeze-thaw cycles (for repeated sample preparation) and stable at ambient temperature for at least eight hours (the maximum duration for sample preparation). If the first analytical run is invalid, the measured sample should be stable for a second injection. Analytes must remain stable in the matrix at the intended storage temperature and duration of the study.

[0159] An analyte is considered stable in one of the above tests when the average concentration of the analyte in at least three analytical QC samples does not increase or decrease by more than 15% at low, medium, and high concentrations.

[0160] Description of the experiment

[0161] Verify operation

[0162] Three valid validation runs were conducted over three different days. Each run consisted of two calibration curves (one at the beginning and one at the end of the validation run), two double-blank samples, two blank samples, and 35 QC samples at five concentration levels. QC levels included LLOQ (10 pg / ml), QC... 低 (25pg / ml), QC 中 (100pg / ml), QC 高 ULOQ (1000 pg / ml) and ULOQ (10000 pg / ml) concentration levels. After analyzing the ULOQ samples, two double-blank samples were directly measured to determine the residues of the method.

[0163] Selective I

[0164] During the validation run, double-blank and blank human plasma samples from seven different subjects were processed and analyzed.

[0165] Selective II

[0166] Seven blank plasma samples from different subjects were incorporated into the analyte under LLOQ for processing and analysis. The intra-assay accuracy and precision of the samples were evaluated based on two calibration curves (one measured at the start of the validation run and one at the end of the validation run).

[0167] Recovery rate and matrix effect

[0168] To determine the recovery rate from human plasma, the peak area of ​​the treated QC samples (spiked samples before extraction) was compared with the peak area of ​​the treated blank plasma samples (supernatant) containing nominal analyte concentrations of QC Low, QC Medium, QC High, and QCULOQ (spiked samples after extraction). The peak area found in the spiked supernatant corresponded to 100% recovery and was compared with the corresponding peak areas of the spiked and treated plasma samples.

[0169] The matrix effect of at least six different batches was determined by calculating the ratio of the peak area in the presence of matrix (measured by analyzing blank plasma after extraction and incorporation of analyte) to the peak area in the absence of matrix (using water instead of plasma). This assay was performed at low, medium, high, and ULOQ levels.

[0170] Stability test

[0171] Reinjection reproducibility

[0172] Repeat the analysis of processed and measured Cal and QC samples (prepared in human plasma) from the valid run. Reinject after overnight storage at 10°C (autosampler) and after 1 week storage at -20°C. Check the run against the acceptance criteria of the validation run. Compare the calculated average of the QC samples between the initial run and the reinjection run.

[0173] Tabletop stability test

[0174] Seven samples from each LLOQ, QC Low, QC Medium, QC High, and ULOQ group of human plasma were thawed at ambient temperature and held at that temperature for 8 hours. The samples were then processed and analyzed. Concentration values ​​in the “short-term” samples were compared to those in the newly processed QC samples. Concentrations were calculated based on two newly prepared CAL groups measured at the start and end of the validation run.

[0175] Freezing / thawing stability test

[0176] Seven samples from each LLOQ, QC Low, QC Medium, QC High, and ULOQ group of human plasma were stored at approximately -20°C for at least 24 hours and thawed without assisted thawing at ambient temperature. Upon complete thawing, the samples were refreezed under the same conditions for at least 12 hours. This freeze-thaw cycle was repeated twice. After the third cycle, the samples were processed and analyzed. The concentrations in the frozen and thawed samples were compared to those in the newly processed QC samples. Concentrations were calculated based on two newly prepared CALs measured at the start of the validation run and another CAL measured at the end.

[0177] Method Application

[0178] To verify the applicability of the developed method, the concentrations of LSD and OH-LSD in plasma samples from three healthy volunteers who received a single oral dose of 5 μg. This corresponds to a very low LSD dose used in clinical trials of microdose administration (Holze et al., 2021a). The study was conducted in accordance with the Declaration of Helsinki and approved by the Medical Ethics Committee of the Academic Hospital of Maastricht and Maastricht University. The use of LSD in humans was authorized by the Netherlands Anti-Drug Agency. All volunteers provided written informed consent prior to participation in the study. To establish concentration-time curves, blood samples were collected in heparin-coated tubes at the following time points: 0, 0.5, 1, 1.5, 2, 3, 4, and 6 days post-treatment. Blood samples were centrifuged, and plasma was frozen at -20°C until analysis.

[0179] Results of method validation and application

[0180] A sensitive LC-MS / MS method was developed and fully validated using a simple and rapid sample analysis workflow.

[0181] Method Validation

[0182] Validation run: Method linearity, accuracy, and precision

[0183] LSD

[0184] All calibration curves from the three validation runs were valid (Table 6). All calibration curves were linear, with correlation coefficients ≥ 0.997. Figure 3 (A-3C). During the validation run, a total of 105 QC samples were analyzed. Of these 105 QC samples, 100 met the QC sample specifications (Table 8).

[0185] OH-LSD

[0186] All calibration curves from the three validation runs were valid (Table 7). All calibration curves were linear, and the correlation coefficients for all runs were ≥0.997. Figure 3 (A-3C). During the validation run, a total of 105 QC samples were analyzed. Of these 105 QC samples, 99 met the QC sample specifications (Table 9).

[0187] Table 6: Accuracy and Precision Data of LSD Calibration Curves

[0188]

[0189] *Apart from the range of 85%-115% (80%-120% for LLOQ), it is not used for calculation.

[0190] Table 7: Accuracy and Precision Data of OH-LSD Calibration Curves

[0191]

[0192] Figure 3 A-3C are calibration curves for LSD and OH-LSD in human plasma. Linearity was observed in the concentration range of 10 to 10,000 pg / ml, with a high correlation coefficient of ≥0.997. Analysis was performed on July 18 (A), 20 (B), and 21 (C), 2020. The developed method achieved a limit of quantitation of 10 pg / ml and exhibited a linear relationship between analyte signal and concentrations from 10 to 10,000 pg / ml.

[0193] Table 8: Intra- and Inter-measurement Accuracy and Precision of LSD

[0194]

[0195] *Apart from the range of 85%-115% (80%-120% for LLOQ), it is not used for calculation.

[0196] Table 9: Intra- and Inter-assay Precision and Accuracy of OH-LSD Determination

[0197]

[0198] *Apart from the range of 85%-115% (80%-120% for LLOQ), it is not used for calculation.

[0199] Selective

[0200] Selective I

[0201] Processed double-blank human plasma from seven different subjects showed no significant interference with the analyte (≤12.1%) (Table 10). Selectivity was also evaluated in the presence of deuterated ISTD (blank sample). ISTD did cause insignificant interference with LSD (≤15.3%) and slight interference with OH-LSD (≤25.4%). Importantly, the interference observed in the plasma from different subjects was consistent. Overall, the method is selective for the analyte studied, such as… Figure 4 As shown.

[0202] Table 10: Selectivity of LSD and OH-LSD in human plasma

[0203]

[0204] Figure 4 A-4D demonstrates the selectivity of LSD and OH-LSD in human plasma. Seven OH-LSDs are shown. Figure 4 A) and LSD Figure 4 B) Overlay of double blank (thick black line) and LLOQ (dashed line) chromatograms. Seven OH-LSD ( Figure 4 C) and LSD Figure 4 D) Overlay of blank (thick black line) and LLOQ (dashed line) chromatograms. The interference from the human plasma matrix is ​​negligible compared to the lower limit of quantitation (LLOQ) signals obtained with LSD and OH-LSD.

[0205] Figure 4 A-4D demonstrates the selectivity of LSD and OH-LSD in human plasma. Seven OH-LSDs are shown. Figure 4 A) and LSD Figure 4 B) Overlay of double blank (gray) and LLOQ (blue-green) chromatograms. Seven OH-LSD ( Figure 4 C) and LSD Figure 4 D) Overlay of blank (gray) and LLOQ (blue-green) chromatograms. The interference from the human plasma matrix is ​​negligible compared to the lower limit of quantitation (LLOQ) signals obtained with LSD and OH-LSD.

[0206] Selective II

[0207] All samples met the Selectivity II specification (accuracy: 82.2%–100%, accuracy for plasma 1–7: ≤6.29%), highlighting the selectivity and sensitivity of this method in analyzing LSD and OH-LSD concentrations up to 10 pg / ml in plasma. Results for LSD and OH-LSD are shown in Table 11.

[0208] Table 11: Selectivity II of LSD and OH-LSD in human plasma

[0209]

[0210] Residue

[0211] The residual between the two injections was ≤0.1%. Two double-blank samples were measured directly after injection of the ULOQ sample. The average signal intensity of the second double-blank sample accounted for 19.6% and 14.7% of the average LSD and OH-LSD signals at the LLOQ level, respectively (Table 12).

[0212] Table 12: Residuals of LSD and OH-LSD between different injections

[0213]

[0214] Recovery rate

[0215] Tables 13 and 14 list the total recoveries of LSD and OH-LSD, respectively. The recoveries of all analytes were consistent across the entire concentration range and between plasma samples from different subjects. The mean recoveries of LSD and OH-LSD were calculated to be 98.3% ± 1.35% and 102% ± 3.84%, respectively. Compared with LSD and OH-LSD, ISTD, LSD-d3, and OH-LSD-d... 10 The recovery rates are similar.

[0216] Table 13: Recovery rates of LSD from human plasma of seven individuals

[0217]

[0218] Table 14: Recovery rates of OH-LSD from human plasma of seven individuals

[0219]

[0220] Matrix effect

[0221] The matrix effects of LSD and LSD-d3 are shown in Table 15. The mean matrix effect of LSD was +8%, and that of LSD-d3 was +18%. The matrix effect was consistent across different plasma batches (%CV ≤ 5.77%) and was independent of the LSD concentration used (25–10,000 pg / mL: ≤ 5.53%).

[0222] Table 15: Matrix effects of LSD and LSD-d3 in human plasma of seven individuals

[0223]

[0224] The matrix effects of OH-LSD and OH-LSD-d10 are shown in Table 16. The average matrix effect of LSD is -10%, and for OH-LSD-d10... 10 The value was -6.8%. The matrix effect was consistent across different plasma batches (%CV ≤ 5.77%) and was independent of the concentration of OH-LSD used (CV% 25-10000 pg / mL: ≤ 2.65%).

[0225] Table 16: OH-LSD and OH-LSD-d in human plasma of seven individuals 10 matrix effect

[0226]

[0227] Stability test

[0228] Reinjection reproducibility

[0229] The validation run and its re-injection were valid. This indicates that the run can be re-injected after overnight storage at 10°C in an autosampler, and after at least one week storage at -20°C in the event of LC-MS / MS system failure. After overnight storage at 10°C, the deviation of the mean QC values ​​between the two runs was between -0.451% and +2.3% for LSD and between -1.59% and +2.08% for OH-LSD. The re-injected QC samples met the specification criteria for the validation run. The results are presented in Tables 17 and 18. After 8 days at -20°C, the deviation of the mean QC values ​​between the two runs was between -1.85% and +1.02% for LSD and between -2.09% and +1.9% for OH-LSD. The re-injected QC samples met the specification criteria for the validation run. The results are presented in Tables 19 and 20.

[0230] Table 17: QC results from re-injected LSD after overnight storage at 10°C in an autosampler.

[0231]

[0232] Table 18: QC results from re-injected OH-LSD after overnight storage at 10°C in an autosampler.

[0233]

[0234] Table 19: QC results from re-injected LSD after 8 days of storage at -20°C.

[0235]

[0236] Table 20: QC results from re-injected OH-LSD after 8 days of storage at -20°C.

[0237]

[0238] Freezing / thawing and short-term stability

[0239] After three freeze / thaw cycles and eight hours at room temperature, the plasma concentrations of LSD and OH-LSD did not change significantly (Tables 21-24). After three freeze / thaw cycles, the plasma concentration change of LSD was ≤8.83%, and that of OH-LSD was ≤6.46%. After 8 hours of storage at room temperature, the plasma concentration changes of LSD and OH-LSD were ≤3.81% and ≤4.52%, respectively.

[0240] Table 21: Stability of LSD after three freeze-thaw cycles

[0241]

[0242] Table 22: Stability of OH-LSD after three freeze-thaw cycles

[0243]

[0244] Table 23: Stability of LSD after 8 hours of storage at room temperature

[0245]

[0246] Table 24: Stability of OH-LSD after 8 hours of storage at room temperature

[0247]

[0248] Clinical application of LC-MS / MS method

[0249] The application of this method was evaluated by analyzing the pharmacokinetic (PK) levels of LSD and OH-LSD in three healthy volunteers treated with an oral dose of 5 μg of LSD base. Figure 5 Three healthy volunteers were administered an oral dose of 5 μg of LSD base in ethanol (Holze et al., 2021). Plasma concentrations of LSD and OH-LSD were quantified before treatment and for up to six hours after treatment. Figure 5 Concentration-time curves for LSD and OH-LSD are shown. Mean and standard deviation are displayed.

[0250] The mean peak plasma levels for LSD and OH-LSD were 178 pg / ml (SD: 30.6 pg / ml) and 10.4 pg / ml (SD: 2.59 pg / ml), respectively. LSD reached T0.05 approximately 1 hour after treatment. maxOH-LSD peaked after 3 hours. The LSD concentration measured after only a 5 μg dose was approximately 7 to 18 times higher than the method's limit of quantitation. Therefore, even for very low, so-called micro-dose doses, the pharmacokinetic (PK) of LSD can be directly determined (Kuypers et al., 2019). In the case of OH-LSD, a larger volume of plasma sample is required to determine the plasma concentration-time curve after a 5 μg dose. Three times more plasma (150 μl instead of 50 μl) was used, and extraction was performed as described above, with three times more acetonitrile used for extraction. Sensitivity was improved by evaporating the extract and reconstructing the residue in a 150 μl mixture of mobile phase A and mobile phase B (9 / 1 v / v). This example demonstrates that the sensitivity of the method can be easily improved by using a larger volume of sample. Future considerations will include injecting a larger volume of extract, which is retained and concentrated on the capture column in the first step. In the second step, the flow direction is reversed, allowing the sample to be loaded and eluted on the analytical column. This column switching procedure will improve sensitivity if the sample can be retained on the capture column. Importantly, this avoids the time-consuming solvent evaporation step.

[0251] Overall, the application examples demonstrate that this method is suitable for quantifying clinical samples using micro-dose LSD. Furthermore, the method can be easily adjusted if the sensitivity of the analysis needs to be improved.

[0252] in conclusion

[0253] Compared to other bioanalytical methods for measuring LSD in human plasma, the method described herein requires only small sample sizes and is characterized by a simple extraction procedure, which facilitates efficient analysis. The extraction protocol results in near-complete analyte recovery. Minimal matrix effects were observed in different plasma batches, and the matrix did not interfere with the analysis of either LSD or OH-LSD. Within the selected calibration range, the quantification of both analytes was accurate and precise, and comparable to levels observed in humans who have received LSD. Overall, this bioanalytical method is an important tool for further developing LSD as a therapeutic agent.

[0254] Throughout this application, all publications, including U.S. patents (if any), are cited by author, year, and patent number. The full citations of these publications are listed below. The disclosures of these publications and patents, in their entirety, are incorporated herein by reference to provide a more comprehensive description of the current state of the art to which this invention pertains.

[0255] The invention has been described by way of example, and it should be understood that the terminology used is intended to be descriptive rather than restrictive.

[0256] It is obvious that many modifications and variations of the present invention can be made based on the above-described content. Therefore, it should be understood that the invention can be practiced in ways other than those specifically described within the scope of the appended claims.

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Claims

1. A method for measuring and identifying lysergic acid diethylamide, abbreviated as LSD, and its major metabolite 2,3-dihydro-3-hydroxy-2-oxoergot diethylamine, abbreviated as OH-LSD, the method comprising the following steps: 300 µL of plasma obtained from the individual will be used as a sample; as well as The LSD and OH-LSD in the sample were measured, identified, and quantified by LC-MS / MS analysis, including: taking 50 µL aliquots from the sample, mixing them with the internal standard working solution, centrifuging, injecting the supernatant into the LC-MS / MS system, online dilution via a T-connector installed in front of the analytical column, selecting a pH-tolerant analytical column to use a pH of 9.0 for mobile phase A, wherein the analysis run time for each sample was 4 minutes, and the LC-MS / MS analysis step had a limit of quantification of 0.5 pg LSD.

2. The method of claim 1, wherein the method has an extraction time of 40 minutes for every 96 samples.

3. The method of claim 1, wherein the method is performed after administration of a microdose of LSD, and wherein the method is capable of measuring, identifying and quantifying the LSD up to six hours after administration.

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