Journal of Chromatography B, 976–977 (2015) 78–83

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Simultaneous determination of remimazolam and its carboxylic acid metabolite in human plasma using ultra-performance liquid chromatography–tandem mass spectrometry Ying Zhou, Hongyun Wang, Ji Jiang, Pei Hu ∗ Clinical Pharmacology Research Center, Peking Union Medical College Hospital, Peking Union Medical College and Chinese Academy of Medical Sciences, Beijing 100730, China

a r t i c l e

i n f o

Article history: Received 14 August 2014 Accepted 22 November 2014 Available online 2 December 2014 Keywords: Determination Remimazolam Metabolite Ultra-performance liquid chromatography–tandem mass spectrometry Human plasma

a b s t r a c t A robust and validated method based on ultra-performance liquid chromatography coupled with tandem mass spectrometry (UPLC–MS/MS) has been developed for the simultaneous determination of remimazolam, which is a new chemical entity, and its major carboxylic acid metabolite (M1) in human plasma. Plasma samples were pre-purified by protein precipitation procedure and analyzed using an isocratic chromatographic separation over an Acquity UPLC CSH C18 column. The mobile phase consisted of acetonitrile–water containing 10 mM ammonium formate and 0.1% formic acid at a flow rate of 0.4 mL min−1 . Positive electrospray ionization was employed as the ionization source in the multiple reaction monitoring (MRM) mode. The analysis time was about 1.5 min. The method was fully validated over the concentration range of 0.5–1000 ng mL−1 for both analytes. The lower limit of quantification (LLOQ) was 0.5 ng mL−1 . Inter- and intra-batch precision was less than 8.4% and the accuracy was within 88.8–107.0%. The mean extraction recoveries obtained from three concentrations of QC plasma samples were 96.8%, 98.7% and 98.6% for remimazolam, 98.7%, 99.8% and 101.5% for M1, respectively. Selectivity, matrix effect and stability were also validated. The method was applied to the pharmacokinetic study of remimazolam in Chinese healthy subjects. © 2014 Elsevier B.V. All rights reserved.

1. Introduction Remimazolam is a short-acting GABA (A) receptor agonist that can be used as an intravenous sedative agent for potential use in day-case procedures, and the induction and maintenance of anesthesia [1,2]. As a member of the benzodiazepine class of drugs, remimazolam is designed to undergo rapid hydrolysis in the body by nonspecific tissue esterases to its pharmacologically inactive carboxylic acid metabolite (M1). Preclinical studies in sheep demonstrated that remimazolam produced a more rapid onset of action, and a shorter duration of action, compared with midazolam, which compatible with its potential human use as a short-acting i.v. sedative [3]. Also, a phase IIa clinical trial evaluating the procedural sedative effect for upper GI endoscopy in patients shows that the time to recovery from sedation was shorter and more consistent with remimazolam, compared to midazolam [2]. Therefore, because of its organ-independent metabolism and fast-acting onset

∗ Corresponding author. Tel.: +86 10 6915 6576; fax: +86 10 6915 8365. E-mail address: [email protected] (P. Hu). http://dx.doi.org/10.1016/j.jchromb.2014.11.022 1570-0232/© 2014 Elsevier B.V. All rights reserved.

and recovery, remimazolam appears to have potential advantages compared to other currently available short-acting sedatives. Remimazolam Tosilate (Fig. 1(a)) was approved by China Food and Drug Administration as an investigational new drug for the potential use in day-case procedures, and the induction and maintenance of anesthesia in 2013 and is currently being evaluated in phase I trials. Remimazolam Tosilate is designed to undergo rapid hydrolysis in the body by nonspecific tissue esterases to its major metabolite M1. As we known, it is necessary to know about the exposure of the main metabolites considering their safety in human based on the FDA guidelines for industry safety testing of drug metabolites [4]. However, currently, few references are available about the determination of remimazolam or its main metabolites, except for one paper [5], that had mentioned an HPLC–MS/MS method to determine the concentration of remimazolam and its main metabolites in human plasma. The method described in this paper had less sensitivity, needed longer total run time, and required much larger plasma volume for sample preparation. Herein, we developed and fully validated a simple, sensitive and rapid method for simultaneous determination of remimazolam and its main metabolite (M1) in human plasma using only 50 ␮L

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Fig. 1. Chemical structures of remimazolam Tosilate (a), M1 (b), [13 C2 ] remimazolam (c), and [13 C2 ] M1 (d).

plasma for each sample preparation. The run time was 1.5 min for each sample analysis, and the use of a stable isotope of the analyte as the internal standard (IS) was thought to be able to yield better assay performance results [6]. The objective of this study was to establish and validate an UPLC–MS/MS method with high sensitivity, accuracy and specificity for the simultaneous determination of remimazolam and its main metabolite in human plasma and to support the pharmacokinetic studies of remimazolam in Chinese healthy subjects. 2. Experimental 2.1. Chemicals and solvents Remimazolam Tosilate and its internal standard [13 C2 ] Remimazolam (Fig. 1(c)), carboxylic acid metabolite of remimazolam (M1, Fig. 1(b)) and its internal standard [13 C2 ] M1 (Fig. 1(d)) were provided by Jiangsu Hengrui Medicine Co., Ltd. (Jiangsu, China). Methanol and acetonitrile was of chromatographic grade and obtained from Burdick & Jackson Lab. Ammonium formate and formic acid were of analytical grade and purchased from Sigma–Aldrich Co. LLC and Sinopharm Chemical Reagent Co., Ltd., respectively. Drug-free human plasma (anticoagulant: Heparin Sodium) was obtained from six different healthy subjects who were drug-free for at least two weeks. Distilled water was prepared with a Milli-Q water purifying system. 2.2. Calibration standard (CS) and quality control (QC) samples in human plasma Stock solutions of remimazolam and M1 for CS and QC were prepared separately in methanol to a final concentration of 1 mg mL−1

for each analyte and a mixed stock solution of IS (10 ␮g mL−1 ) was also prepared in methanol. These stock solutions were further diluted to yield working solutions at several concentration levels. All the solutions were stored at −30 ◦ C and brought to room temperature (25 ◦ C) before use. Calibration standards and QC samples in plasma were prepared by diluting corresponding working solutions with drug-free human plasma, respectively. The final concentrations of calibration standards were 0.5, 2, 5, 20, 50, 200, 500 and 1000 ng mL−1 . The final concentrations of QC samples for the evaluation of intra- and inter-batch precision and accuracy, recovery and matrix effect and stability were 1.5, 30 and 750 ng mL−1 . Mixed IS working solution (100 ng mL−1 ) was prepared with methanol. Plasma samples were stored at −70 ◦ C. 2.3. Sample preparation All the plasma samples were pretreated by protein precipitation procedure. Plasma sample (50 ␮L) was spiked with 150 ␮L of IS solution (100 ng mL−1 ). The mixture was vortex-mixed for 3 min and centrifuged at 16,242 × g for 5 min, and then 150 ␮L of the supernatant were collected and evaporated to dryness under a stream of nitrogen at 40 ◦ C. The residues were reconstituted in 100 ␮L of acetonitrile/water containing 10 mM ammonium formate and 0.1% formic acid (40:60, v/v) and vortex-mixed for 1 min and then centrifuged at 16,242 × g for 1 min before injection. Finally, 7.5 ␮L of the dissolved sample was injected to the UPLC–MS/MS system. 2.4. Liquid chromatography–tandem mass spectrometry Plasma samples were analyzed using Acquity UPLC Core system coupled with Xevo-TQS triple quadrupole mass spectrometer

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Y. Zhou et al. / J. Chromatogr. B 976–977 (2015) 78–83

Fig. 2. Product ion spectra of remimazolam (a), M1 (b), [13 C2 ] remimazolam (c) and [13 C2 ] M1 (d).

equipped with an electrospray ionization (ESI) source in the positive mode (Waters Co., MA, USA). Chromatographic separation was carried out on an Acquity UPLC CSH C18 (50 mm × 2.1 mm, 1.7 ␮m) column at 25 ◦ C. The autosampler temperature was 10 ◦ C. The mobile phase was acetonitrile/water containing 10 mM ammonium formate and 0.1% formic acid (40:60, v/v). The flow rate was 0.4 mL min−1 and the run time was 1.5 min with an injection volume of 7.5 ␮L. Mass spectrometer was operated in the multiple reaction monitoring (MRM) mode. The MRM transitions of remimazolam and its IS were m/z 439 → 362 and m/z 444 → 412, M1 and its IS were m/z 425 → 407 and m/z 430 → 412, respectively. The ionization source conditions were capillary voltage 3 kV, cone voltage 45 V, source temperature 150 ◦ C and desolvation temperature 600 ◦ C. The optimized collision energy of remimazolam and its IS were both 30 V, M1 and its IS were 20 V and 18 V, respectively. The cone and desolvation gas flow rates were 150 and 1000 L h−1 , respectively. Data acquisition and processing were performed using MassLynx software (version 4.1). 2.5. Method validation 2.5.1. Selectivity, linearity and LLOQ The method was validated for selectivity, sensitivity, linearity, recovery, matrix effects, precision, accuracy and stability according to the US Food and Drug Administration (FDA) [7] and China Food and Drug Administration (CFDA) guidelines [8] for the validation of bioanalytical methods. The selectivity of the assay was investigated by analyzing six lots of analyte-free plasma. Endogenous interferences were observed in different lots of plasma sample, and the “cross-talk” between MRM transitions was evaluated by analyzing the different blank

plasma sample, which was only spiked with remimazolam, M1 at a concentration of upper limit of quantitation (1000 ng mL−1 ) or the mixed IS of 100 ng mL−1 . Calibration standards in human plasma were prepared and analyzed in three independent runs. The calibration curves were constructed by weighted (1/x2 ) least-square linear regression analysis of the peak area ratio of analyte to its internal standard against nominal analyte concentration. The LLOQ is defined as the lowest concentration on the calibration curve. The deviations of back-calculated concentrations of calibration standards from their nominal values should be within ±20% for LLOQ and ±15% for all other calibration levels.

2.5.2. Recovery and matrix effects The extraction recovery of remimazolam and M1 were calculated at three levels (1.5, 30 and 750 ng mL−1 ) by comparing two groups of control samples: (A) analyte spiked to plasma and prepared normally (pre-extraction); (B) analyte spiked after extraction of blank plasma (post-extraction). The ratio (A/B × 100) is defined as the extraction recovery. The reproducibility of the extraction procedure was determined as RSD%. The matrix effects were evaluated at three levels (1.5, 30 and 750 ng mL−1 ) too. Two groups of samples were prepared: group 1 was prepared in plasma originating from six different subjects and submitted to the sample preparation process and spiked with analyte after processing (B); group 2 was prepared to evaluate the MS/MS response for a pure standard of analyte dissolved in the mobile phase (C). The ratio (B/C × 100) is defined as the absolute matrix effects. The assessment of the relative matrix effects, which was expressed as RSD%, was made by a direct comparison of B values between six different lots of plasma. The inter-subject

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Fig. 3. Representative MRM chromatograms of remimazolam, M1, [13 C2 ] remimazolam, and [13 C2 ] M1: (1) blank plasma sample, (2) blank plasma sample spiked with two analytes at LLOQ and two internal standards (100 ng mL−1 ), and (3) plasma sample collected from a healthy subject at 2 h after 1-min IV administration of 0.075 mg kg−1 Remimazolam Tosilate injection.

variability of matrix effects at each concentration level should be less than 15% [9–11]. 2.5.3. Accuracy and precision Intra- and inter-batch precision and accuracy were determined by measuring the concentrations of analyte in plasma in five replicates of QC samples at three different concentrations for three separate batches. 2.5.4. Stability The stabilities of remimazolam and M1 in biological matrices and in working solution at different storage conditions were evaluated as follows: the short-term stability of analytes in human plasma was assessed after 4 h of storage at room temperature. The long-term stabilities of analytes in human plasma and in working solution were assessed after 33 days of storage in a freezer at −70 ◦ C and 44 days of storage in a freezer at −30 ◦ C, respectively. The stability of analytes was assessed after three freeze–thaw cycles (−70 ◦ C to room temperature). The stability of analytes in extracts was also tested after 48 h at 10 ◦ C. 2.6. Pharmacokinetic study The validated method was applied to determine plasma concentrations of remimazolam and M1 for Chinese Phase I single dose study. This was a single-center, double-blinded, randomized, single ascending-dose study of remimazolam administered as a 1-min IV injection, compared with midazolam (0.075 mg kg−1 ). Up to 64 Chinese healthy subjects were planned for enrollment in up to 10 cohorts (doses ranging from 0.01 mg kg−1 to 0.35 mg kg−1 ). Plasma samples were collected at specific time points and stored at −70 ◦ C before analysis. The study was approved by the Ethics Committee of Peking Union Medical College Hospital, and all subjects signed the Informed Consent Form before the study.

In order to avoid the “cross-talk”, we chose m/z 444 and m/z 430 as the precursor ions for [13 C2 ] remimazolam and [13 C2 ] M1, respectively. As we known, it is better that the mass difference is higher than 3 Da in the synthesis of isotope internal standard. However, in our study, the number of 13 C is two and the compounds contain bromine, which is the cause of “cross-talk”. Well, fortunately, although these precursor ions having only about 20% of the abundance of that of the molecular ions, all of these were improved in the present study and have no interference with each other. Also, we chose the product ions based on the most abundant peak, cleaner chromatogram and higher S/N ratio. In the meanwhile, the ionization and fragmentation parameters such as capillary voltage, cone voltage, source temperature, desolvation temperature and collision energy were optimized to obtain the most stable and the highest signal response. MRM scan mode was selected to ensure high specificity of this method. Negative ionization was also applied, but compared to the positive mode, the sensitivity was much lower. Fig. 2 shows the product ion spectra of remimazolam, M1 and their internal standards. The choice of mobile phase and an appropriate elution program are critical because the properties of the mobile phase primarily govern the separation of the analytes, the degree of analyte ionization and the good robustness of the method. In this study, acetonitrile was chosen as the organic phase because acetonitrile revealed higher mass spectrometric response and lower background than methanol. Besides, additional formic acid and buffer solution had increased the S/N ratios and improved peak shape. Finally, When acetonitrile–water containing 10 mM ammonium formate and 0.1% formic acid (40:60, v/v) pumped at a flow rate of 0.4 mL min−1 at 25 ◦ C was used as eluent, a good chromatographic profile and sensitivity was achieved. After careful comparison of several columns, such as, a Varian Pursuit Diphenyl column (50 mm × 2.0 mm, 3 ␮m), a Phenomenex Luna PFP column (50 mm × 2.0 mm, 3 ␮m) and an Acquity UPLC CSH C18 column (50 mm × 2.1 mm, 1.7 ␮m). Finally, the CSH C18 column was proved

3. Result and discussion 3.1. UPLC–MS/MS optimization An UPLC–MS/MS method for the detection of remimazolam and M1 in human plasma was developed. It has been demonstrated that the selection of MRM transition ion pairs and the optimization of ESI parameters are crucial for a selective and sensitive method. In this work, the protonated molecular ions [M+H]+ were chosen as the precursor ions for all analytes. Although, the most abundant molecular ion peaks of [13 C2 ] remimazolam and [13 C2 ] M1 respectively are peaks corresponding to m/z 441 (or m/z 443, isotopic peak of bromine) and m/z 427 (or m/z 429, isotopic peak of bromine), there are overlap region between those peaks and molecular ion peaks of remimazolam and M1, which can cause “cross-talk” effect.

Table 1 Results of extraction recovery and matrix effects of remimazolam and M1 in human plasma (n = 6). Compound

Nominal concentration (ng mL−1 )

Remimazolam

1.50 30.0 750

96.8 (4.8) 98.7 (3.9) 98.6 (8.8)

102.5 (6.4) 91.5 (6.3) 87.8 (7.2)

M1

1.50 30.0 750

98.7 (8.8) 99.8 (7.3) 101.5 (8.3)

129.2 (8.7) 126.7 (4.1) 119.1 (3.6)

a

Expressed as RSD %.

Recovery% (RSD%)

Matrix effects% (inter-subject variability%)a

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Table 2 Accuracy and precision of intra- and inter-batch for the detection of remimazolam and M1 in human plasma (n = 5). Compound

Nominal concentration (ng mL−1 )

Intra-batch accuracy (%)

Inter-batch accuracy (%)

Intra-batch precision (RSD%)

Inter-batch precision (RSD%)

Remimazolam

1.50 30.0 750

94.7 107.0 91.6

94.0 96.0 88.8

3.1 3.1 1.2

2.9 8.4 2.2

M1

1.50 30.0 750

94.7 94.3 92.7

94.7 93.3 97.2

2.0 2.8 0.7

3.7 6.2 3.7

to be more suitable as it can provide sufficient retention and better peak shape for analytes.

3.2. Validation procedure 3.2.1. Selectivity, linearity and LLOQ The representative chromatograms of the blank plasma, blank plasma spiked with two analytes and two internal standards and a subject’s plasma sample obtained 2 h after 1-min IV administration of 0.075 mg kg−1 Remimazolam Tosilate injection are shown in Fig. 3. No peaks interfering were observed at the retention times of remimazolam, M1 and the internal standards in the MRM chromatograms of blank samples from 6 lots of human plasma. In addition, the “cross-talk” between MRM transitions was evaluated. Representative chromatograms clearly showed little interference at the retention time of remimazolam, M1 and the internal standards. Although IS of remimazolam had response in the MRM transition of remimazolam, it did not affect the quantitation of remimazolam because the interference peak area from IS was lower

than 20% of that from LLOQ sample of remimazolam, which met the FDA guidelines (not shown in the figure). A calibration curve was established ranging from 0.5 to 1000 ng mL−1 for plasma. Calibration curve was regressed using linear equation with a weighting factor of 1/x2 . Coefficients of correlation of all calibration curves were more than 0.99. The LLOQ for all analytes was 0.5 ng mL−1 in plasma. The deviations of back-calculated concentrations of calibration standards from their nominal values were between 5.8% and 13.4% for LLOQ, and between −4.0% and 5.0% for all other calibration levels. 3.2.2. Recovery and matrix effects The observed recovery for the extraction method from plasma (mean value and RSD%, n = 6) is shown in Table 1. The mean extraction recoveries obtained from three concentrations of QC plasma samples were 96.8%, 98.7% and 98.6% for remimazolam, 98.7%, 99.8% and 101.5% for M1, respectively. Matrix effects and inter-subject variability data from three concentrations of QC plasma samples were also summarized in Table 1. The inter-subject variabilities were no more than 8.7% for all

Fig. 4. Mean (±SD) plasma concentration–time curves of remimazolam and M1 in 6 subjects after a single-dose IV administration of 0.075 mg kg−1 Remimazolam Tosilate injection.

Y. Zhou et al. / J. Chromatogr. B 976–977 (2015) 78–83 Table 3 Results of stability of remimazolam and M1 under different storage conditions (n = 5). Storage condition

Nominal concentration (ng mL−1 )

Measured mean concentration (RE%) M1 (ng mL−1 )

1.50 30.0 750

1.55 (3.3) 29.5 (−1.7) 825 (10.1)

1.48 (−1.5) 30.1 (0.3) 745 (−0.6)

Long-term stabilityb

1.50 30.0 750

1.39 (−7.2) 26.8 (−10.7) 746 (−0.6)

1.40 (−6.4) 28.1 (−6.3) 705 (−6.0)

Freeze–thaw stabilityc

1.50 30.0 750

1.60 (6.4) 29.9 (−0.4) 815 (8.7)

1.54 (2.5) 30.8 (2.5) 735 (−2.1)

Auto-sampler stabilityd

1.50 30.0 750

1.55 (3.2) 29.4 (−2.1) 823 (9.7)

1.69 (12.5) 32.9 (9.8) 784 (4.6)

a

c d

1-min IV administration of Remimazolam Tosilate injection to Chinese healthy subjects. The mean plasma concentration–time curve for the 0.075 mg kg−1 Remimazolam injection dose (n = 6) is shown in Fig. 4. 4. Conclusion

Remimazolam (ng mL−1 ) Short-term stabilitya

b

83

Stored at room temperature (25 ◦ C) for 4 h. Stored at −70 ◦ C for 33 days. After three freeze–thaw cycles. Kept at 10 ◦ C for 48 h.

analytes. This indicated little or no difference in ionization efficiency of remimazolam and M1 from different plasma lots. 3.2.3. Accuracy and precision Precision and accuracy values were determined on three different batches by measuring five replicates of QC samples at three concentration levels in each batch. The results are listed in Table 2. All the observed data for the intra- and inter-batch precision were less than 8.4%, and accuracy were between 88.8% and 107.0%. These data indicated that the method provides adequate precision and accuracy for the determination of two analytes. 3.2.4. Stability The stability tests of the analytes were designed to cover expected conditions of handling of clinical samples. The stability of the analytes in human plasma was investigated under a variety of storage and processing conditions and they were found stable under the following conditions: at room temperature (25 ◦ C) for 4 h; at 10 ◦ C for 48 h in autosampler post-extraction; after 3 freeze–thaw cycles (−70 ◦ C to room temperature); at −70 ◦ C for 33 days. Working solutions of remimazolam and M1 stored at −30 ◦ C for 44 days also showed good stability. The stability results are summarized in Table 3. 3.3. Application of the method in pharmacokinetic studies In the present study, a specific, sensitive and rapid method based on UPLC–MS/MS was developed and fully validated to simultaneously quantify remimazolam and its carboxylic acid metabolite M1 in human plasma. This method was successfully applied to determine the pharmacokinetic profile of remimazolam after

A rapid, sensitive and specific UPLC–MS/MS method coupled with protein precipitation has been developed and validated for the simultaneous determination of remimazolam and its carboxylic acid metabolite M1 in human plasma. The challenges in the development of the method were the high sensitivity required following low doses, the specificity of the method, and the selection of MRM transition ion pairs of the internal standards. All of these were optimized in the present study. The procedure was fully validated. This method was successfully applied to determine remimazolam and M1 in human plasma and characterize the pharmacokinetic profile of remimazolam in Chinese healthy subjects. Acknowledgments This study was financially supported by Jiangsu Hengrui Medicine Co., Ltd., and we thank them for providing us with remimazolam, M1 and their isotopic internal standards. This work was also supported by a grant from the National Program on Key Research Project of New Drug Innovation (No. 2012ZX09303006002). References [1] G.J. Kilpatrick, M.S. Mclntyre, R.F. Cox, J.A. Stafford, G.J. Pacofsky, G.G. Lovell, R.P. Wiard, P.L. Feldman, H. Collins, B.L. Waszczak, G.S. Tilbrook, CNS 7056: a novel ultra-short-acting Benzodiazepine, Anesthesiology 1 (2007) 60–66. [2] W.K. Rogers, T.S. McDowell, Remimazolam, a short-acting GABA(A) receptor agonist for intravenous sedation and/or anesthesia in day-case surgical and non-surgical procedures, IDrugs 12 (2010) 929–937. [3] R.N. Upton, A.A. Somogyi, A.M. Martinez, J. Colvill, C. Grant, Pharmacokinetics and pharmacodynamics of the short-acting sedative CNS 7056 in sheep, Br. J. Anaesth. 6 (2010) 798–809. [4] Food and Drug Administration, Guidance for Industry Safety Testing of Drug Metabolites, FDA, Rockville, MD, USA, 2008. [5] L.J. Antonik, D.R. Goldwater, G.J. Kilpatrick, G.S. Tilbrook, K.M. Borkett, A placebo- and midazolam-controlled phase I single ascending-dose study evaluating the safety, pharmacokinetics, and pharmacodynamics of remimazolam (CNS 7056): Part I. Safety, efficacy, and basic pharmacokinetics, Anesth. Analg. 2 (2012) 274–283. [6] E. Stokvis, H. Rosing, J.H. Beijnen, Stable isotopically labeled internal standards in quantitative bioanalysis using liquid chromatography/mass spectrometry: necessity or not? Rapid Commun. Mass Spectrom. 3 (2005) 401–407. [7] Guidance for Industry Bioanalytical Method Validation, 2001, Available http://www.fda.gov/downloads/Drugs/GuidanceComplianceRegulatory at: Information/Guidances/ucm070107.pdf [8] China Food and Drug Administration, 2005. Available at: http://www.sfda. gov.cn/directory/web/WS01/images/u6Rp9KpzuB2bSy0qm0+ravwabRp9HQvr +8vMr11ri1vNSt1PIucGRm.pdf [9] P.J. Taylor, Matrix effects: the Achilles heel of quantitative high-performance liquid chromatography–electrospray-tandem mass spectrometry, Clin. Biochem. 38 (2005) 328–334. [10] B.K. Matuszewski, M.L. Constanzer, C.M. Chavez-Eng, Strategies for the assessment of matrix effect in quantitative bioanalytical methods based on HPLC–MS/MS, Anal. Chem. 75 (2003) 3019–3030. [11] B.K. Matuszewski, Standard line slopes as a measure of a relative matrix effect in quantitative HPLC–MS bioanalysis, J. Chromatogr. B 830 (2006) 293–300.

Simultaneous determination of remimazolam and its carboxylic acid metabolite in human plasma using ultra-performance liquid chromatography-tandem mass spectrometry.

A robust and validated method based on ultra-performance liquid chromatography coupled with tandem mass spectrometry (UPLC-MS/MS) has been developed f...
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