A method for the bioanalysis of ipratropium bromide in plasma

Abstract

The application belongs to the technical field of biological analysis, and particularly relates to a biological analysis method of ipratropium bromide in blood plasma. The method comprises the following steps: detecting ipratropium bromide in blood plasma by using liquid chromatography-mass spectrometry, wherein the liquid chromatography conditions are as follows: using a C18 column as a chromatographic column, using a methanol solution as mobile phase B, gradient elution, and column temperature being 25 DEG C to 35 DEG C; and the mass spectrometry uses an ESI ion source and a positive ion detection mode, and ion source parameters are as follows: source temperature being 600 DEG C, gas curtain gas pressure being 35 psi, ion spray voltage being 5500 V, atomization gas pressure being 65 psi, auxiliary gas pressure being 70 psi, and collision gas pressure being 9 psi. The method is a biological analysis method which is simple in operation, high in accuracy, rapid in sensitivity, and strong in specificity, and can promote the quality improvement of the drug aerosol and the reasonable clinical application.

Description

Technical Field

[0001] This invention belongs to the field of bioanalytical technology, specifically relating to a bioanalytical method for ipratropium bromide in plasma. Background Technology

[0002] Chronic obstructive airway disease (mainly referring to chronic obstructive pulmonary disease, COPD) is the third leading cause of death after cardiovascular disease and cancer.

[0003] Ipratropium bromide inhalation aerosol, with ipratropium bromide as its main active ingredient, is a first-line drug for the prevention and treatment of dyspnea associated with chronic obstructive airway disease. Although many domestic companies now produce ipratropium bromide inhalation aerosol, meeting some clinical needs, the original drug still holds advantages in quality control and clinical data accumulation. With the continuous emergence of generic drugs, the requirements for their quality consistency are becoming increasingly stringent. Therefore, there is an urgent need to establish a bioanalytical system aligned with international standards to provide strong support for the quality evaluation and clinical application of generic drugs. In the research and evaluation of generic drugs, to understand their pharmacokinetic characteristics, bioavailability, and safety, it is essential to accurately measure the concentration of ipratropium bromide in biological samples such as plasma and urine of subjects or patients.

[0004] Furthermore, after inhalation, only a small amount of this drug is absorbed into the systemic circulation; the majority is metabolized into inactive metabolites in the liver and primarily excreted through the kidneys. This aerosol inhaler (20 μg / puff) is rapidly absorbed after inhalation, taking effect in approximately 5 minutes. It exerts a high local concentration in the airways to dilate the bronchi. The estimated systemic bioavailability after inhalation is less than 10% of the administered dose, and the absorbed blood concentration is extremely low (pg level). To meet the requirements for human pharmacokinetic and bioequivalence studies of ipratropium bromide inhaler (20 μg / puff × 1 puff), the lower limit of quantitation must be at least 1 pg / mL or lower.

[0005] However, existing technologies lack sufficiently documented efficient methods for detecting ipratropium bromide concentrations in human plasma, particularly quantitative analytical methods with complete bioanalytical methodological validation. Furthermore, given the characteristics of inhalation administration and the drug's high airway selectivity, conventional HPLC and existing mass spectrometry methods have low detection sensitivity (LLOQ > 8 ng / mL). Current methods employ complex solid-phase extraction for pretreatment, resulting in high consumable consumption and increased manpower, drastically increasing research costs and significantly burdening clinical trials. In addition, traditional analytical methods have long processing times, exceeding 5 minutes per injection for a single sample, making them unsuitable for the high-throughput testing requirements of large-scale clinical studies (over 4000 samples per study), directly extending the research cycle. More importantly, the lower limit of quantitation (LOQ) fails to meet the clinical research requirements of inhaled formulations due to their high airway selectivity and low systemic exposure, making accurate analysis of large batches of samples difficult in clinical studies, thus greatly hindering the development and clinical application of ipratropium bromide inhalation aerosols.

[0006] Therefore, there is an urgent need to develop a simple, accurate, rapid, sensitive and specific bioanalytical method to promote the improvement of the quality of the drug's aerosol formulation and its rational clinical application. Summary of the Invention

[0007] To address the aforementioned technical problems, the present invention aims to provide a bioanalytical method for ipratropium bromide in plasma.

[0008] The present invention adopts the following technical solution: A bioanalytical method for ipratropium bromide in plasma, the method comprising the following steps: detecting ipratropium bromide in plasma using liquid chromatography-mass spectrometry, wherein the liquid chromatography conditions are: using a C18 column as the chromatographic column, using an aqueous solution containing 0.1% formic acid and 10 mM ammonium formate as mobile phase A, using methanol solution as mobile phase B, gradient elution, and a column temperature of 25℃~35℃; The mass spectrometer uses an ESI ion source in positive ion detection mode. The ion source parameters are as follows: source temperature 600℃, curtain gas pressure 35psi, ion spray voltage 5500V, nebulizer gas pressure 65psi, auxiliary gas pressure 70psi, and collision gas pressure 9psi.

[0009] In some implementations, the gradient elution procedure is as follows: .

[0010] In some embodiments, the liquid chromatography is performed at a flow rate of 0.43-0.47 mL / min.

[0011] In some embodiments, the C18 column is of model ACQUITY HSS T3 and has a specification of 1.8µm x 2.1 x 75 mm.

[0012] In some embodiments, the mass spectrometry conditions include a mass spectrometry acquisition time of 3.5 min; the mass spectrometry parameters of ipratropium bromide are: ion pair (mother ion / daughter ion) 332.2 / 166.2, residence time 200.0 ms, declustering voltage 80.0 V, collision energy 36.0 eV, inlet voltage 10.0 V, and collision chamber outlet voltage 10.0 V; the mass spectrometry parameters of its internal standard ipratropium bromide-d3 are: ion pair (mother ion / daughter ion) 335.2 / 169.1, residence time 200.0 ms, declustering voltage 80.0 V, collision energy 36.0 eV, inlet voltage 10.0 V, and collision chamber outlet voltage 10.0 V.

[0013] In some embodiments, the minimum amount of plasma used is 200 μL.

[0014] In some embodiments, the lower limit of quantification for ipratropium bromide is 0.5 pg / mL.

[0015] In some embodiments, before sampling and detection using liquid chromatography-tandem mass spectrometry, the sample is pretreated. The pretreatment method is as follows: an internal standard working solution is added to the plasma sample, mixed thoroughly, and then acetonitrile solution is added for protein precipitation. After mixing, the sample is centrifuged, the supernatant is collected, dried under nitrogen, and reconstituted to obtain the test sample. The internal standard working solution is a 20.00 pg / mL ipratropium bromide-d3 acetonitrile-methanol solution.

[0016] In some implementations, the plasma sample is vortexed before the internal standard working solution is added.

[0017] In some embodiments, the resolution solvent is an acetonitrile solution.

[0018] Compared with the prior art, the present invention has the following advantages: 1. The detection method described in this invention is currently the only disclosed method for detecting ipratropium bromide concentration in human plasma. It uses UHPLC-MS / MS method, which has high analytical efficiency and can be used to support non-clinical and clinical research on this drug. 2. High specificity: It adopts LC-MS / MS multiple reaction monitoring mode to effectively eliminate interference from endogenous substances, and combines deuterated internal standard to correct matrix effect; 3. Simple operation: The protein precipitation method is used for pretreatment, which is simple and convenient. It can minimize the matrix effect, enhance the robustness of the method, and is suitable for high-throughput sample analysis. The extracted sample is easy to preserve and has good stability. 4. Rapid analysis: The detection method described in this invention has a short time, with a single-needle injection time of 3.5 minutes, which can quickly and efficiently meet the high-throughput clinical testing needs and greatly improve the testing efficiency; 5. High sensitivity: With a plasma sample volume of only 200µL, the limit of quantitation can reach 0.5 pg / mL, which can meet the detection requirements of extremely low blood drug concentrations in clinical studies of ipratropium bromide inhalation formulations. This can reduce the dosage and whole blood collection volume of subjects, which is beneficial to increasing the safety of clinical studies and subject compliance, and meets the requirements of clinical pharmacokinetic studies containing this compound; moreover, the linear range is reasonably selected, which can accurately determine the concentration of the analyte compound in plasma. 6. Accurate and reliable: The methodology has been fully validated (precision, accuracy, recovery, matrix effect, stability, etc.) and meets the relevant guidelines for quantitative analysis of biological samples. Attached Figure Description

[0019] Figure 1 The chromatogram of the ipratropium bromide pure solution reference sample under quantitative ion pair conditions at M / Z 332.2→166.2 (retention time of target compound 1.59 min). Figure 2 The chromatogram of the ipratropium bromide pure solution reference sample under quantitative ion pair conditions at M / Z 332.2→124.1 (retention time of target compound 1.60 min). Figure 3 The chromatogram corresponding to the use of Ultimate UHPLC AQ-C18 (75 mm × 2.1 mm, particle size 1.8 μm) column in Example 2 (retention time of target compound 1.80 min). Figure 4 The chromatogram corresponding to the use of ACQUITY UPLC® HSS T3 (75 mm × 2.1 mm, particle size 1.8 μm) column in Example 2 (retention time of target compound 1.50 min). Figure 5 The chromatogram corresponding to the use of Shim-pack GIST C18-AQ HP (2.1×100 mm, particle size 3.0 μm) column in Example 2 (retention time of target compound 1.84 min). Figure 6 The chromatogram for the mobile phase A in Example 2 is 10 mM ammonium formate (containing 0.1% formic acid), and the mobile phase B is methanol (retention time of the target compound is 1.50 min). Figure 7 The chromatogram for Example 2 is as follows: mobile phase A is 0.1% acetic acid water and mobile phase B is methanol (retention time of target compound is 1.44 min). Figure 8 The chromatogram for mobile phase A being 10 mM ammonium formate and mobile phase B being methanol in Example 2 is shown (retention time of target compound is 1.53 min). Figure 9 The chromatogram for Example 2 is as follows: mobile phase A is 1 mM ammonium formate (containing 0.1% formic acid) and mobile phase B is methanol (retention time of target compound is 1.52 min). Figure 10 The chromatogram for Example 2 is as follows: mobile phase A is 5 mM ammonium formate (containing 0.1% formic acid) and mobile phase B is methanol (retention time of target compound is 1.51 min). Figure 11 The chromatogram for the target compound retention time is 1.50 min when mobile phase A is 20 mM ammonium formate (containing 0.1% formic acid) and mobile phase B is methanol in Example 2. Figure 12 The chromatogram corresponding to gradient 1 in Example 2 (retention time of target compound 1.43 min). Figure 13 The chromatogram corresponding to gradient 2 in Example 2 (retention time of target compound 1.54 min); Figure 14 The chromatogram corresponding to gradient 3 in Example 2 (retention time of target compound 1.61 min). Figure 15 This is a pharmacokinetic curve of the subjects in Example 5; Figure 16 This is a diagram showing the daughter and parent ions of ipratropium bromide. Figure 17 This is a diagram showing the daughter and parent ions of ipratropium bromide-d3. Figure 18 The MRM chromatogram of ipratropium bromide in a blank matrix is ​​shown. Figure 19 The MRM chromatogram of ipratropium bromide-d3 in the blank matrix is ​​shown. Figure 20 The MRM chromatogram of ipratropium bromide in the sample at the lower limit of quantification; Figure 21 The MRM chromatogram is shown for ipratropium bromide-d3 in the sample at the lower limit of quantification. Detailed Implementation

[0020] The present invention will be further described in detail below with reference to the specific embodiments and accompanying drawings. The scope of protection of the present invention is not limited to the following embodiments. Variations and advantages that can be conceived by those skilled in the art without departing from the spirit and scope of the inventive concept are included in the present invention and are protected by the appended claims. The processes, conditions, reagents, experimental methods, etc., for implementing the present invention, except as specifically mentioned below, are all common knowledge and general knowledge in the art, and the present invention does not have any particular limitations.

[0021] In this invention, unless otherwise stated, percentages and ratios mentioned in the reagents are weight percentages and weight ratios.

[0022] In this invention, the unit "M" represents moles per liter (mol / L), and "v / v" and "v / v / v / v" represent volume ratios.

[0023] Sample sources: Ipratropium bromide (purity 95.5%) and internal standard ipratropium bromide-d3 (purity 99.4%), methanol (HPLC grade), acetonitrile (HPLC grade), and isopropanol (HPLC grade) were purchased from Sigma-Aldrich (USA), and formic acid (HPLC grade) and ammonium formate (AR) were purchased from Sinopharm Group. Deionized water (18.2 mΩ, TOC ≤ 50 ppb) was prepared using a laboratory ultrapure water system. Blank plasma and whole blood were collected from hospitals and approved by the Clinical Trial Medical Research Ethics Committee.

[0024] Healthy subjects (n=6) underwent a fasting test and were given ipratropium bromide inhalation aerosol (Atrovent®) at a dose of 20 μg / puff × 1 puff. Venous blood was collected at 18 time points per cycle, including 0 h before administration (within 90 min before administration) and 2 min (0.033 h), 5 min (0.083 h), 7 min (0.117 h), 10 min (0.167 h), 15 min (0.25 h), 20 min (0.333 h), 25 min (0.417 h), 30 min (0.5 h), 45 min (0.75 h), 1 h, 2 h, 3 h, 4 h, 6 h, 8 h, 12 h, and 24 h after administration. Each time, 4 mL of blood was collected.

[0025] Approximately 4 mL of whole blood was collected each time into a blood collection tube containing EDTA-K2 anticoagulant. Within 90 minutes of collection, the blood sample was centrifuged at approximately 4°C (1700 g × 10 min). After centrifugation, the plasma sample was divided into two aliquots into labeled cryovials. ≥1 mL of the separated plasma was transferred to a test tube, and the remaining plasma was transferred to a backup tube. The time from sample collection to transfer to the freezer should not exceed 3 hours. Centrifuged plasma samples were stored directly at -60°C or transferred from -20°C to -60°C within 12 hours. Sample pretreatment must be performed entirely at room temperature under white light.

[0026] Standard and control samples are prepared by adding the working solution of the analyte to blank plasma.

[0027] Example 1: Screening of mass spectrometry conditions The parent ion [M+H]+ was found to be 332.2 in Q1 MS mode, and its fragment ions 166.2 and 124.1 were subsequently obtained in Product Ion mode. These ion pairs were imported into the multiple reaction monitoring (MRM) scan mode list, and parameters such as collision energy were systematically optimized. The optimization results showed that 166.2 had the highest abundance among all fragment ions and exhibited no significant interference. Figure 1-2 As shown, therefore, 332.2→166.2 was ultimately selected as the final quantitative ion pair in this experiment.

[0028] Meanwhile, by gradually adjusting the source temperature from room temperature to 600℃, the signal intensity changes of the target compound were monitored. The results showed that when the source temperature reached 600℃, the ionization efficiency and ion transport efficiency of the target compound significantly improved, and the signal intensity reached its maximum and remained stable. Furthermore, by gradually optimizing various gas parameters and monitoring the signal intensity and stability of the target compound, the following parameters were ultimately determined to provide the optimal signal intensity and good reproducibility for the target compound: Curtain Gas (CUR) of 35.0 psi, Collision Gas (CAD) of 9.0 psi, Atomizing Gas (Gas1) of 65.0 psi, and Assist Gas (Gas2) of 70.0 psi (acceptable range 65.0 psi–75.0 psi).

[0029] Example 2 Screening of liquid chromatography conditions The optimization mainly focuses on several aspects, including the chromatographic column, mobile phase, flow rate, and gradient elution.

[0030] 1.1 Column Screening Ipratropium bromide is a quaternary ammonium salt compound with strong polarity and water solubility. While hydrophilic columns (such as HILIC) exhibit strong retention for highly polar compounds, their forward elution gradient can easily lead to instability in the ion source spray, thus affecting the sensitivity of mass spectrometry detection. Therefore, a reversed-phase nonpolar column is preferable for separation. This example compares three different brands and models of columns: Ultimate UHPLC AQ-C18 (75 mm × 2.1 mm, particle size 1.8 μm), ACQUITY UPLC® HSS T3 (75 mm × 2.1 mm, particle size 1.8 μm), and Shim-pack GIST C18-AQHP (2.1 × 100 mm, particle size 3.0 μm). Other parameters were: mobile phase A: 0.1% formic acid in water, mobile phase B: methanol, conventional gradient elution program, column temperature 30℃, and flow rate 0.30 mL / min. The results showed that, for the analyte ipratropium bromide, as Figure 3-5 As shown, the ACQUITY UPLC® HSS T3 column exhibits the best separation performance. This column demonstrates excellent compatibility with highly aqueous mobile phases and features special end-capping treatment that effectively reduces the activity of silanol groups while enhancing interactions with polar compounds, thereby significantly improving the retention capacity of ipratropium bromide and effectively separating the analyte from impurity peaks.

[0031] 1.2 Mobile phase screening The choice of mobile phase has a significant impact on the peak height and shape of compounds. An ACQUITY UPLC® HSST3, 1.8 μm, 2.1*75 mm, was used with a standard gradient elution program, column temperature 30℃, and flow rate 0.30 mL / min. Methanol was initially used as the organic phase to screen for different aqueous phases.

[0032] Aqueous phase screening: Ammonium formate, formic acid, and acetic acid were used as aqueous phases to investigate their effects on the separation of the target compound and the response values. Figure 6-8 As shown, the separation degree and peak shape of the target compound are optimal when using 10 mM ammonium formate (containing 0.1% formic acid) as the aqueous phase. This aqueous phase system provides a stable pH environment, improves peak shape and retention time repeatability, and enhances the retention of the target compound through weak ion interactions, making it more conducive to its effective separation from impurities.

[0033] Adding a low concentration of electrolyte to the aqueous phase increases the response of most compounds and weakens the matrix effect due to the increased conductivity and surface area / volume ratio of the electrospray droplets. However, the response decreases with increasing concentration. The mechanism is as follows: the addition of electrolyte reduces the droplet radius, which helps form gaseous ions. However, the rapid droplet shrinkage caused by the high-temperature atomizing gas reduces the time for compound ions to distribute to the droplet surface. At the same time, the increased concentration of electrolyte ions within the droplet makes the gradually shrinking droplet more crowded, hindering the migration of compound ions to the droplet surface, ultimately leading to a decrease in the compound response. Therefore, comparative studies were conducted on adding different concentrations of electrolyte (e.g., 1 mM ammonium formate, 5 mM ammonium formate, 10 mM ammonium formate, 20 mM ammonium formate) to a 0.1% formic acid mobile phase. The results demonstrate that only the specific concentration range of this invention (e.g., 5–10 mM) can ensure good signal strength while avoiding ion suppression. Furthermore, under aqueous phase conditions of 10mM ammonium formate (containing 0.1% formic acid), it can meet the concentration point of 0.5pg / mL for LLOQ and the signal-to-noise ratio is >5.

[0034] Adding a low concentration of electrolyte to the aqueous phase enhances the response of most compounds and weakens the matrix effect due to the increased conductivity of the electrospray droplets and the increased surface area / volume ratio. However, the response of compounds decreases when the electrolyte concentration is further increased. This invention investigated the response changes when different concentrations of electrolyte (ammonium formate) were added to a mobile phase containing 0.1% formic acid, with concentrations of 1 mM, 5 mM, 10 mM, and 20 mM. The comparative results show that only within the specific concentration range of 5–10 mM defined in this invention can ion suppression be effectively avoided while maintaining good signal strength. Furthermore, under aqueous phase conditions containing 10 mM ammonium formate and 0.1% formic acid, the detection requirement of a lower limit of quantitation (LLOQ) of 0.5 pg / mL can be met, with a signal-to-noise ratio greater than 5.

[0035] Furthermore, when 10 mM ammonium formate (containing 0.1% formic acid) is used as the aqueous phase, it can effectively shield the residual negative charge on the stationary phase surface, reduce tailing, and make the chromatographic peaks sharper. Narrow and high peaks directly improve the signal-to-noise ratio (S / N), thus allowing for a lower limit of quantitation.

[0036] Organic phase screening: Methanol and acetonitrile were used as the organic phases, respectively, and their separation effects were compared. Experimental results showed that methanol provided the best separation degree and peak shape for the target compound.

[0037] 1.3 Optimization of mobile phase gradient and flow velocity We optimized the flow rate and investigated the chromatographic behavior at 0.3 mL / min, 0.4 mL / min, 0.45 mL / min, and 0.5 mL / min. The results showed that at a flow rate of 0.45 mL / min, the column pressure was moderate, the elution time was suitable, the peak shape was sharp and symmetrical, and the resolution met the requirements for quantitative analysis.

[0038] Based on the selected chromatographic column ACQUITY UPLC® HSS T3, 1.8μm, 2.1*75mm, and mobile phase A: 10mM ammonium formate (containing 0.1% formic acid), mobile phase B: methanol, column temperature 30℃, and flow rate 0.45 mL / min, the elution program was screened.

[0039] Meanwhile, the gradient elution program was optimized by adding a gentle gradient before the high-proportion organic phase wash to achieve effective separation of impurity peaks from the target peak. After screening, the optimal mobile phase ratio and gradient elution conditions were finally determined to be gradient 3, which can complete the baseline separation of all components within 3.5 min, with symmetrical and sharp peaks, smooth peak shapes, and suitable retention times, meeting the analytical requirements for accurate quantification.

[0040] Table 1 Gradient 1

[0041] Table 2 Gradient 2

[0042] Table 3 Gradient 3

[0043] Example 3: Sample Pretreatment Method To investigate the extraction efficiency of different precipitants, 200.0 μL of each of the mixed low, medium, and high concentration quality control samples were added to a 96-well plate, along with 50.0 μL of internal standard working solution. Subsequently, methanol, acetonitrile, 0.2% formic acid methanol, and 0.2% formic acid acetonitrile were added to each sample for protein precipitation. Protein precipitation, centrifugation, nitrogen drying, and redissolution / dilution were performed according to the established pretreatment procedures. Simultaneously, spiked samples were prepared: the analyte and internal standard were added to the extracted blank matrix to maintain the same concentration as the low, medium, and high concentration quality control samples, respectively, for calculating the extraction recovery rate. The results showed that acetonitrile exhibited the best precipitation effect, with recoveries consistently around 65% at both low and high concentrations. Therefore, acetonitrile was selected as the protein precipitant for subsequent experiments.

[0044] Example 4: Methodological Validation Example 1. Solution and sample preparation Standard series samples: Accurately weigh appropriate amounts of each reference standard (ipratropium bromide), place them in brown glass bottles, and after correction factor adjustment, accurately measure a certain volume of methanol to dissolve and shake well to prepare stock solutions S1 and S2 with an ipratropium bromide concentration of approximately 1.000 mg / mL. Accurately pipette appropriate amounts of each stock solution and dilute stepwise with methanol:water (50:50, v:v) to obtain standard curve working solutions. Finally, prepare standard curve plasma samples (STDs) using blank plasma, with ipratropium bromide concentrations ranging from 0.5000 to 60.00 pg / mL.

[0045] Quality control samples (QCs): Five concentration levels of diosgenin were prepared using methods similar to those used for the standard series samples; the lower limit of quantitation was 0.5000 pg / mL, the low quality control (LQC) concentration was 1.500 pg / mL, the medium quality control 1 (MQC1) concentration was 5.000 pg / mL, the medium quality control 2 (MQC2) concentration was 20.00 pg / mL, and the high quality control (HQC) concentration was 45.00 pg / mL.

[0046] Internal standard working solution: Calculate the mass of ipratropium bromide-d3 reference standard after correction by the correction factor, dissolve it in a certain volume of methanol and shake well to prepare an internal standard stock solution with a concentration of approximately 1.000 mg / mL. Accurately pipette an appropriate amount of each of the above internal standard stock solutions, dilute with acetonitrile to obtain an internal standard working solution with a concentration of 20.00 ng / mL for ipratropium bromide-d3.

[0047] 2. Plasma pretreatment steps: (1) Use a pipette to add 200.0 μL of sample (standard curve sample, quality control sample, test sample, equilibration sample, etc.) to the corresponding position in the 96-well plate; (2) Add 50.0 μL of internal standard working solution (concentration: 20.00 pg / mL) to each sample. (3) Add 550 μL of acetonitrile solution to precipitate the protein, and shake in a 96-well plate mixer for 5 min to mix thoroughly. (4) Centrifuge the sample for 10 min (5℃, 2480g); (5) Take 500 μL of supernatant into a 96-well plate and dry it with nitrogen; (6) Add 100 μL of acetonitrile:ultrapure water (10:90, v:v) to reconstitute.

[0048] 3. Chromatographic and mass spectrometric conditions Table 4 Chromatographic conditions

[0049] Table 5 Mass Spectrometry Conditions

[0050] 4. Methodological Validation The method was validated according to the guidelines of the 2020 edition of Chinese Pharmacopoeia Part 4, 9012 and ICH M10, including stability, selectivity, linearity, accuracy, precision, recovery, and matrix effects.

[0051] Selective Samples prepared from six different sources of blank plasma, one source of blank high-lipid matrix, and one source of blank hemolyzed matrix were processed and then injected for analysis. The peak area of ​​interfering substances at the analyte elution position must be less than 20% of the analyte peak area and less than 5% of the internal standard peak area at the quantitation limit.

[0052] Standard curve A linear regression equation (weighting factor W = 1 / x²) was calculated using the theoretical concentration of the analyte as the x-axis and the peak area ratio of the analyte to the internal standard as the y-axis. Method validation involved two-sample analysis of each analytical batch against the standard curve.

[0053] Precision and accuracy Method validation involved measuring six samples from five concentration control samples in each analytical batch. For the limit of quantitation (LOQ), intra- and inter-batch precision (calculated as relative standard deviation (RSD)) was acceptable if less than 20%, and accuracy (calculated as relative deviation (RE)) was acceptable if between -20% and 20%. For all other concentration levels of QC samples, intra- and inter-batch precision for each component was acceptable if less than 15%, and accuracy was acceptable if between -15% and 15%.

[0054] stability To investigate the stability of each analyte in plasma samples, LQC and HQC were placed in different temperatures and environments. After placement, six samples were analyzed. A total of six placement conditions were investigated: 24 hours of white light exposure at room temperature, 192 hours of placement at 5℃ after preparation, five freeze-thaw cycles at -80℃ (from -80℃ to room temperature), three freeze-thaw cycles at -20℃ (from -20℃ to room temperature), 7 days of placement at -80℃, and 7 days of placement at -20℃.

[0055] Recovery rate Take 200 μL of blank plasma, extract it (without adding internal standard working solution), add the analyte solution and internal standard working solution to make the final concentration the same as LQC, MQC, and HQC, and inject for analysis. Separately extract 6 aliquots each of LQC, MQC, and HQC, and inject for analysis. Calculate the extraction recovery rate based on the peak area ratio of the two processing methods.

[0056] Matrix effect Six blank plasma samples from different sources were collected, extracted (without internal standard working solution), and then mixed with analyte solution and internal standard working solution of the same concentration as those used in LQC and HQC. The mixture was then vortexed before measurement. Water was used in place of plasma and processed according to the same method. The matrix factor was calculated using the ratio of peak areas obtained by the two methods. The matrix effect was assessed by the RSD of the matrix factor normalized to internal standard; a value less than 15% was considered acceptable.

[0057] Results and Discussion Method selectivity like Figures 3 to 4 As shown, the retention times of ipratropium bromide and ipratropium bromide-d3 are approximately 1.50 and 1.51 min, respectively, with no co-eluent interference peaks at the retention times.

[0058] Standard curve The linear range for determining ipratropium bromide in plasma samples from clinical studies was 0.5000–60.00 pg / mL. The typical linear regression equation for the standard curve of the analyte was: ipratropium bromide: y = 5.65 x + 0.0811.

[0059] Detection limit The limit of quantitation (LOQ) for ipratropium bromide in the sample was 0.5000 pg / mL, with a signal-to-noise ratio (SNR) of 9.2. Based on an SNR of 3, the limit of detection (LOD) was calculated to be 0.1630 pg / mL.

[0060] Precision and accuracy of the method The precision and accuracy results both met the acceptance criteria, and the results are shown in Table 6. Table 6 shows the precision and accuracy data for the determination of ipratropium bromide in human plasma.

[0061] Table 6. Precision and accuracy data for ipratropium bromide

[0062] Recovery rate The recovery rate was calculated by comparing the peak areas of the prepared samples (LQC, MQC, and HQC) containing the analyte and internal standard after pretreatment with those of the prepared samples containing the analyte and internal standard without pretreatment.

[0063] calculate:

[0064] LQC, MQC and HQC concentration levels: The extraction recoveries of ipratropium bromide were 65.5%, 64.2% and 65.4%, respectively; the overall recovery was 65.0% and the overall variation CV was 1.11%; the consistency was good.

[0065] Matrix effect The matrix factors of ipratropium bromide at LQC and HQC concentration levels, after internal standard normalization, were 103.0% and 103.0%, respectively, with RSDs of 7.77% and 2.91%, respectively. These results indicate that the matrix effect does not interfere with the accuracy of analyte analysis.

[0066] Plasma stability study The results of the plasma stability test are shown in Table 7. The results show that ipratropium bromide is stable under the tested conditions. Table 7 shows the stability data of ipratropium bromide in human plasma (n=6).

[0067] Table 7. Stability data of ipratropium bromide in human plasma

[0068] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the technical principles of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

[0069] Example 5: In vivo pharmacokinetic study The validated method was used to analyze plasma ipratropium bromide concentrations to evaluate the pharmacokinetic characteristics of ipratropium bromide inhalation aerosol (Atrovent®, dosage: 20 μg / puff × 1 puff (calculated as C20H30BrNO3), batch number: F67371; product batch number: 301902. Content: 0.371 mg / g; administration method: oral inhalation, once per cycle (20 μg / puff × 1 puff)). Plasma concentrations of ipratropium bromide at different sampling times were determined using LC-MS / MS. The pharmacokinetic behavior of ipratropium bromide aerosol in humans was studied, and the sensitivity of the detection method met the requirements for experimental detection of ipratropium bromide. The pharmacokinetic curves are shown below. Figure 15 As shown in the figure. This demonstrates that this method can also be applied to pharmacokinetic studies in generic drug development.

Claims

1. A bioanalytical method for ipratropium bromide in plasma, characterized in that, The method includes the following steps: detecting ipratropium bromide in plasma using liquid chromatography-mass spectrometry (LC-MS), wherein the LC conditions are: using a C18 column as the chromatographic column, using an aqueous solution containing 0.1% formic acid and 10 mM ammonium formate as mobile phase A, using methanol solution as mobile phase B, gradient elution, and a column temperature of 25℃~35℃. The mass spectrometer uses an ESI ion source in positive ion detection mode. The ion source parameters are as follows: source temperature 600℃, curtain gas pressure 35psi, ion spray voltage 5500V, nebulizer gas pressure 65psi, auxiliary gas pressure 70psi, and collision gas pressure 9psi.

2. The bioanalytical method for ipratropium bromide in plasma according to claim 1, characterized in that, The gradient elution procedure is as follows: 。 3. The bioanalytical method for ipratropium bromide in plasma according to claim 1, characterized in that, The flow rate used in the liquid chromatography was 0.43-0.47 mL / min.

4. The bioanalytical method for ipratropium bromide in plasma according to claim 1, characterized in that, The C18 column is model ACQUITY HSS T3, with dimensions of 1.8µm and 2.1x75mm.

5. The bioanalytical method for ipratropium bromide in plasma according to claim 1, characterized in that, The mass spectrometry conditions specified include a mass acquisition time of 3.5 min; the mass spectrometry parameters for ipratropium bromide are: ion pair (mother ion / daughter ion) 332.2 / 166.2, residence time 200.0 ms, declustering voltage 80.0 V, collision energy 36.0 eV, inlet voltage 10.0 V, and collision chamber outlet voltage 10.0 V; the mass spectrometry parameters for its internal standard ipratropium bromide-d3 are: ion pair (mother ion / daughter ion) 335.2 / 169.1, residence time 200.0 ms, declustering voltage 80.0 V, collision energy 36.0 eV, inlet voltage 10.0 V, and collision chamber outlet voltage 10.0 V.

6. The bioanalytical method for ipratropium bromide in plasma according to claim 1, characterized in that, The minimum plasma volume used is 200 μL.

7. The bioanalytical method for ipratropium bromide in plasma according to claim 1, characterized in that, The limit of quantification for ipratropium bromide is 0.5 pg / mL.

8. The bioanalytical method for ipratropium bromide in plasma according to claim 1, characterized in that, Before using liquid chromatography-tandem mass spectrometry (LC-MS / MS) for sample injection and detection, the sample must be pretreated. The pretreatment method is as follows: add internal standard working solution to the plasma sample, mix thoroughly, then add acetonitrile solution for protein precipitation, mix well, centrifuge, collect the supernatant, dry under nitrogen, and redissolve to obtain the test sample; the internal standard working solution is a 20.00 pg / mL ipratropium bromide-d3 acetonitrile-methanol solution.

9. The bioanalytical method for ipratropium bromide in plasma according to claim 8, characterized in that, Before adding the internal standard working solution to the plasma sample, the plasma sample is vortexed.

10. The bioanalytical method for ipratropium bromide in plasma according to claim 8, characterized in that, The resolution solvent is an acetonitrile solution.

Images