Development and validation of an LC–MS/MS method for the determination of quetiapine and four related metabolites in human plasma

Pharmacokinetic measurement of the psychotropic compound quetiapine and four related metabolites in human plasma was conducted using a sensitive and specific liquid–chromatography tandem mass spectrometry (LC–MS/MS) assay that has been developed and validated for this purpose. The assay employs a single liquid–liquid extraction of quetiapine and its N-desalkyl (norquetiapine, M211,803, M1), 7-hydroxy (M214,227, M2), 7-hydroxy N-desalkyl (M236,303, M3), and sulfoxide (M213,841, M4) metabolites from human plasma, and utilizes dual-column separation, using Luna C₁₈ columns (50 mm × 2.0 mm, 5 μm) and positive ionization tandem MS detection in the multiple reaction monitoring (MRM) mode of the analytes and their respective stable labeled internal standards. The method provides a linear response from a quantitation range of <0.70 ng/ml to at least 500 ng/ml for each analyte using 40 μl of plasma. The applicable range was extended by dilution up to 100-fold with blank matrix. The accuracy and precision for quetiapine were less than 6.0% and 6.4% for quetiapine, respectively. The accuracy (and precision) was less than 9.4% (5.9%) for norquetiapine; 6.4% (6.2%) for M2; and 10.0% (6.4%) for M3; and 8.6% (9.5%) for M4. This methodology enabled the determination of the pharmacokinetics of quetiapine and its metabolites in human plasma, and an example of its application is presented.     

Development and validation of an HPLC–UV method for the quantification of carbamazepine in rabbit plasma

An isocratic simple rapid assay has been developed and validated for the determination of carbamazepine (CBZ) in both solution form and rabbit plasma using propylparaben as an internal standard. The assay was performed using a μ-Bondapak C₁₈ (150 mm × 4.6 mm i.d) with a mobile phase consisting of methanol and water (50:50), the flow rate was 1 ml/min and UV detection at 285 nm. The method was found to be specific for CBZ, no interfering peaks were observed with an overall analytical run time of 15 min. Accuracy reported as % recovery were found to be 98.37–100.45% and 97.53–103.58% for inter-day and intra-day accuracies, respectively. Inter-day precision (reproducibility) was found to be 0.53–2.75% RSD, while intra-day precision (repeatability) was found to be 1.06–3.7% RSD for the samples studied. The calibration curve was found to be linear with the equation y = 0.2847x + 0.0138, with a correlation coefficient of 0.9999 (R²) over a concentration range of 0.5–40 μg/ml. The limit of quantitation was the lowest concentration. The method is simple and rapid and does not require any preliminary treatment of the sample. The method was fully validated.     

Development and validation of a selective and robust LC-MS/MS method for quantifying trimetazidine in human plasma

AIM： To develop and validate a liquid chromatography-tandem mass spectrometric method （LC-MS/MS） for quantifying trimetazidine in human plasma.METHODS： Sample preparation was based on extracted using acetonitrile only. Chromatography was performed on a C18 analytical column and the retention times were 1.9 and 2.6 min for trimetazidine and cetirizine （internal standard）, respectively. The ionization was optimized using ESI （ ＋ ） and enhanced selectivity was achieved using tandem mass spectrometric analysis via two MRM functions, m/z：267→m/z： 181 and m/z：389→m/z：201 for trimetazidine and cetirizine.[第一段]     

Development and validation of a LC–MS/MS method for determination of bivalirudin in human plasma: Application to a clinical pharmacokinetic study

A simple, sensitive and rapid LC–MS/MS method has been developed and validated for the identification and quantification of bivalirudin in human plasma using nafarelin as the internal standard. Following protein precipitation with methanol, the analytes were separated on a C₁₈ column interfaced with a triple-quadrupole tandem mass spectrometer using positive electrospray ionization. Quantification of bivalirudin was conducted by multiple reaction monitoring (MRM) of the transitions of m/z 1091.4 → (356.4 + 227.4) for bivalirudin and m/z 662.4 → 328.5 for IS. The lower limit of quantification was 1.25 ng/ml, and the assay exhibited a linear range of 1.25–500 ng/ml. The developed assay method was successfully applied to a pharmacokinetic (PK) study in healthy volunteers after intravenous administration of bivalirudin.     

Development and validation of an LC–MS/MS method for quantification of NC-8 in rat plasma and its application to pharmacokinetic studies

ent-16-Oxobeyeran-19-N-methylureido (NC-8) is a recently synthesized derivative of isosteviol that showed anti-hepatitis B virus (HBV) activity by disturbing replication and gene expression of the HBV and by inhibiting the host toll-like receptor 2/nuclear factor-κB signaling pathway. To study its pharmacokinetics as a part of the drug development process, a highly sensitive, rapid, and reliable liquid chromatography tandem mass spectrometry (LC–MS/MS) method was developed and validated for determining NC-8 in rat plasma. After protein precipitation extraction, the chromatographic separation of the analyte and internal standard (IS; diclofenac sodium) was performed on a reverse-phase Luna C₁₈ column coupled with a Quattro Ultima triple quadruple mass spectrometer in the multiple-reaction monitoring mode using the transitions, m/z 347.31 → 75.09 for NC-8 and m/z 295.89 → 214.06 for the IS. The lower limit of quantitation was 0.5 ng/mL. The linear scope of the standard curve was between 0.5 and 500 ng/mL. Both the precision (coefficient of variation; %) and accuracy (relative error; %) were within acceptable criteria of <15%. Recoveries ranged from 104% to 113.4%, and the matrix effects (absolute) were non-significant (CV ≤ 6%). The validated method was successfully applied to investigate the pharmacokinetics of NC-8 in male Sprague–Dawley rats. The present methodology provides an analytical means to better understand the preliminary pharmacokinetics of NC-8 for investigations on further drug development.     

Development and validation of a highly sensitive LC–MS/MS method for the determination of acacetin in human plasma and its application to a protein binding study

A highly sensitive bioanalytical method for the quantification of acacetin in human plasma was developed and comprehensively validated using liquid chromatography-tandem mass spectrometry (LC–MS/MS). A minimal volume of human plasma sample (20 μL) was prepared by simple deproteinization with 80 μL of acetonitrile. Chromatographic separation was performed using Kinetex C₁₈ column with an isocratic mobile phase consisting of water and acetonitrile (20:80, v/v) containing 0.1 % formic acid at a flow rate of 0.3 mL/min over a total run time of 2.0 min. Mass spectrometric detection was performed using multiple reaction-monitoring modes at the mass/charge transitions m/z 285.22 → 242.17 for acacetin and m/z 277.59 → 175.04 for chlorpropamide (internal standard). The calibration curve was linear over the range of 0.1–500 ng/mL with a lower limit of quantitation of 0.1 ng/mL. The coefficients of variation for both intra- and inter-day validation were less than 11.9 %, and the intra- and inter-day accuracy ranged from 96.8 to 108 %. Mean recovery of acacetin in human plasma was within the range of 91.5–95.6 %. This validated LC–MS/MS method was successfully applied to a human plasma protein binding study that indicated extensive and concentration-independent protein binding of acacetin in human plasma.     

LC-MS/MS assays to determine olmesartan concentration in human plasma

LC-MS/MS方法测定人血浆中奥美沙坦@陈英$Institute of Clinical Pharmacology, Central South University!Changsha 410078,Hunan,China @谭志荣$Institute of Clinical Pharmacology, Central South University!Changsha 410078,Hunan,China @周宏灏$Institute of Clinic     

Development and validation of a selective and robust LC-MS/MS method for quantifying ilaprazole and its metabolite ilaprazole sulfone in human plasma

LC-MS/MS方法测定人血浆中艾普拉唑及其代谢产物艾普拉唑砜的浓度@谭志荣$Pharmacogenetics Research Institute, Institute of Clinical Pharmacology, Central South University!Changsha 410078, Hunan, China @张伟$Pharmacogenetics Research Institute, Institute of C     

Development and validation of an LC–ESI–MS/MS method for simultaneous determination of levodopa, dopamine, L-α-methyldopa and 3-O-methyldopa in rat plasma

Parkinson??s disease (PD) is a progressive degenerative disorder of the central nervous system. Levodopa (L-dopa), dopamine precursor is the most effective therapeutic drug for PD patients. Levodopa (LDP) and its three metabolites in rat plasma were determined using high performance liquid chromatography coupled with tandem mass spectrometry (LC?CMS/MS). Method validation was conducted in terms of linearity, accuracy, precision, recovery, specificity, limit of detection, limit of quantification and stability. Correlation coefficients (r ²) were above 0.9965. The intra-day accuracy values at LLOQ (low limit of quantification), LQC (low quality control), MQC (medium quality control) and HQC (high quality control) levels were 85.7?C103.3, 96.5?C105.1, 90.7?C100.1 and 94.2?C101.3?%, respectively. The inter-day accuracy values at LLOQ, LQC, MQC and HQC levels were 77.6?C112.0, 91.1?C109.3, 84.3?C101.0 and 88.2?C103.9?%, respectively. The coefficient of variation (RSD) values of both intra- and inter-day results were within 6.7 and 8.5?%, respectively. The recoveries (mean?±?%SD) for LLOQ, LQC, MQC and HQC were 82.7?±?3.7?C113?±?2.8?%, 86.6?±?5.7?C110.3±3.4?%, 90.9?±?3.6?C106?±?6.5 and 89.7?±?4.5?C97.4?±?6.7?%, respectively. The coefficient of variation (RSD) values of both intra- and inter-day results were within 6.7 and 8.5?%, respectively. The validated LC?CMS/MS method was applied successfully to the measurement of LDP and its metabolites in the rat plasma samples.     

Development and validation of a rapid and sensitive UHPLC–MS/MS method for the determination of paliperidone in beagle dog plasma

In order to evaluate the pharmacokinetic profile of paliperidone extended-release tablets in vivo, a simple and rapid ultra-high performance liquid chromatography–tandem mass spectrometry (UHPLC–MS/MS) method was developed and validated for the determination of paliperidone in beagle dog plasma. Paliperidone and diazepam (internal standard) were extracted from plasma samples with diethyl ether, and then separated on a C₁₈ column (2.1 × 50 mm, 2.6 μm) under gradient elution with methanol–0.1% formic acid at a flow rate of 0.3 ml/min. The compounds were detected using a triple-quadrupole mass spectrometer equipped with an electrospray ionization (ESI) source. The validated method was linear over the concentration range of 1.00–1000.00 ng/ml and the lower limit of quantitation was 1.00 ng/ml. The intra-day and inter-day precision values were not more than 15% (relative standard deviation < 20% at low levels), while the accuracy was within ±10% of nominal values. The validated UHPLC–MS/MS method was successfully applied to an oral pharmacokinetic study of paliperidone extended-release tablets in a beagle dog.     

Bioanalytical method development and validation using incurred samples: Quantitative estimation of metformin in human K3EDTA plasma by LC–MS/MS

Bioanalytical methods should be reproducible and consistent when applying to clinical sample analysis. Incurred sample reanalyses (confirmatory reanalyses) are performed to support clinical data, and regulatory agencies evaluate the same before approval of bioequivalent products/drugs. A confirmatory reanalysis was demonstrated for metformin after administration of 2/500 mg repaglinide + metformin fixed dose formulation under fasted and fed conditions. The liquid chromatography tandem mass spectrometry (LC-MS/MS) method for determination of metformin in human plasma using metformin-d₆as an internal standard has been developed and validated. The ions transitions recorded in multiple reaction monitoring (MRM) were m/z 130.1→60.0 and 136.2→60.0 for metformin and metformin-d₆, respectively. The compounds were isolated by solid phase extraction and separated on a C12 reverse phase (Synergi MAX-RP 80A) column, using isocratic mobile phase flow at a flow rate of 0.8 mL/min. No matrix effect was observed within the linearity range of 10.2–1741.8 ng/mL (r² > 0.99). The acceptable result of confirmatory reanalysis further indicated stability of metformin in the presence of repaglinide. The assay method was found to be highly reproducible and was successfully applied for pharmacokinetic evaluations of metformin in fixed dose combinations with repaglinide.     

Rapid and sensitive LC-MS/MS method for determination of Pravastatin in human plasma

快速灵敏的LC-MS/MS方法检测人血浆中普伐他汀的浓度@张敏$Institute of Clinical Pharmacology, Central South University!Changsha 410078,Hunan,China @谭志荣$Institute of Clinical Pharmacology, Central South University!Changsha 410078,Hunan,China @周宏灏$Ins     

Development of a LC–MS/MS-based method for determining metolazone concentrations in human plasma: Application to a pharmacokinetic study

In this study, a method was developed and validated for the quantification of metolazone in human plasma samples. This method involves high-performance liquid chromatography coupled with tandem mass spectrometry and is more sensitive, selective and rapid than currently available methods. Chromatography was performed using a Phenomenex^® Luna C18 column (100 mm × 2.0 mm, 5 μm, 100 Å) with an isocratic mobile phase of 0.1% formic acid:acetonitrile (40:60, v/v) and zaleplon as an internal standard. The drug and internal standard were extracted by liquid–liquid extraction and analyzed by mass spectrometry in the multiple reaction monitoring mode by using m/z values of 366.20/259.10 for metolazone and 306.20/235.60 for zaleplon. The calibration curve was linear over metolazone concentrations ranging from 0.02 ng/mL to 15 ng/mL. The lower limit of quantification was 0.02 ng/mL. Intra- and inter-assay precisions were 0.9–4.8% and 4.2–6.3%, respectively. The intra- and inter-assay accuracies in quantifying metolazone were 97.5–102.3% and 99.2–104.0%, respectively. Metolazone and zaleplon were eluted within 3.6 minutes, and the retention time was 1.75 minutes for metolazone and zaleplon. The validated method was successfully applied to a pharmacokinetic study of metolazone in human plasma.     

Validated LC–MS/MS method for the determination of sarpogrelate in human plasma: Application to a pharmacokinetic and bioequivalence study in Chinese volunteers

A rapid and sensitive liquid chromatography–tandem mass spectrometry (LC–MS/MS) method was established for the determination of sarpogrelate in human plasma. One-step protein precipitation with acetonitrile was used to extract the analytes from the plasma. Sarpogrelate and tramadol (internal standard, I.S.) were separated on a Venusil MP-C₁₈ column within 1.7 min, using acetonitrile:ammonium acetate (10 mM, pH 6.8) (55:45, v/v) as mobile phase at a flow rate of 1.2 mL/min with an approximately 1:1 split entering the mass spectrometer. Detection was performed on electrospray positive ionization mass spectrometry by multiple reaction monitoring of the transitions of sarpogrelate at m/z 430.3 → 135.3 and of I.S. at m/z 264.1 → 58.0. The assay was validated over the concentration range of 1–1000 ng/mL with a lower limit of quantitation (LLOQ) of 1 ng/mL using 50 μL of plasma. The intra- and inter-day precision (relative standard deviation, R.S.D.) were ≤6.4% and ≤5.4%, respectively, with accuracy (relative error, R.E.) in the range 0.5–3.6%. The method was successfully applied to a pharmacokinetic and bioequivalence study enrolling 22 Chinese volunteers administered sarpogrelate tablets.     

Quantification of the major metabolites of bromhexine in human plasma using RRLC–MS/MS and its application to pharmacokinetics

(E)-4-hydroxydemethylbromhexine (E-4-HDMB) and (E)-3-hydroxydemethylbromhexine (E-3-HDMB) were found as major metabolites, while (Z)-4-hydroxydemethylbromhexine and (Z)-3-hydroxydemethylbromhexine as minor metabolites of bromhexine in human plasma. These compounds were identified in comparison with synthetic authentic samples. A sensitive and selective rapid resolution liquid chromatography tandem mass spectrometry (RRLC–MS/MS) method was developed to quantify the concentration of bromhexine and its two major metabolites (E-4-HDMB and E-3-HDMB) in human plasma. Following solid phase extraction, the analytes were separated on a Zorbax 1.8 μm particle size reversed-phase C₁₈ column, using a gradient elution program with solvents consisting of 0.1% formic acid in acetonitrile and 0.1% formic acid in 5 mM ammonium acetate at a flow rate of 0.7 mL/min. Detection was carried out with an Agilent 6460 triple-quadrupole mass spectrometer operated with an electrospray ionization source mode operated in the positive ion mode. The recovery of bromhexine, E-4-HDMB, E-3-HDMB, and internal standard (IS) was 63.1–70.9%, 60.5–68.4%, 57.0–63.5%, and 87.8%, respectively. The matrix factors of bromhexine, E-4-HDMB, E-3-HDMB, and IS were 89.9–96.7%, 89.6–94.8%, 90.4–91.4%, and 103%, respectively. After an oral administration of 8.0 mg bromhexine to five healthy male subjects, AUC_0–24 h values of bromhexine, E-4-HDMB, and E-3-HDMB were found to be 93.5 ± 31.9, 34.0 ± 14.5, and 15.8 ± 6.89 ng h/mL, respectively; while C_max values were 24.6 ± 5.16, 3.11 ± 1.13, and 5.36 ± 2.55 ng/mL, respectively. Plasma concentration of bromhexine, E-4-HDMB, and E-3-HDMB declined with t_1/2 which gave 3.6 ± 0.5, 8.4 ± 2.7, and 6.4 ± 2.5 h, respectively.     

Development and validation of an EI–GC–MS method for the determination of benzodiazepine drugs and their metabolites in blood: Applications in clinical and forensic toxicology

Benzodiazepines are used widely in daily clinical practice, due to their multiple pharmacological actions. The frequent problems associated with the wide use of benzodiazepines, as well as the multiple incidents of poisonings, led to the necessity for the development of a precise, sensitive and rapid method for the simultaneous determination of the 23 most commonly used benzodiazepines (diazepam, nordiazepam, oxazepam, bromazepam, alprazolam, lorazepam, medazepam, flurazepam, fludiazepam, tetrazepam, chlordiazepoxide, clobazam, midazolam, flunitrazepam, 7-amino-flunitrazepam, triazolam, prazepam, nimetazepam, nitrazepam, temazepam, lormetazepam, clonazepam, camazepam) in blood. A gas chromatographic method combined with mass spectrometric detection was developed, optimized and validated for the determination of the above substances. This method includes liquid–liquid extraction with chloroform at pH 9 and two stages of derivatization using tetramethylammonium hydroxide and propyliodide (propylation), as well as a mixture of triethylamine:propionic anhydride (propionylation). The recoveries were higher than 74% for all the benzodiazepines. The calibration curves were linear within the dynamic range of each benzodiazepine with a correlation coefficient higher than 0.9981. The limits of detection and quantification for each analyte were statistically calculated from the relative calibration curves. Accuracy and precision were also calculated and were found to be less than 8.5% and 11.1%, respectively. The developed method was successfully applied for the investigation of both forensic and clinical toxicological cases of accidental and suicidal poisoning.     

Development of a simultaneous LC–MS/MS method to predict in vivo drug–drug interaction in mice

Cocktail substrates are useful in investigating drug–drug interactions (DDI) that can rapidly identify the cytochrome P450 (CYP) isoforms that interact with test drugs. In this study, we developed and validated five probe drugs for CYP1A, CYP2B, CYP2C, CYP2D, and CYP3A using LC–MS/MS to determine CYP activities in mice. The five probe substrates were caffeine (2 mg/kg), bupropion (30 mg/kg), omeprazole (4 mg/kg), dextromethorphan (40 mg/kg), and midazolam (2 mg/kg) for CYP1A, CYP2B, CYP2C, CYP2D, and CYP3A, respectively. The cocktail substrates were orally administered to male 5-week-old ICR mice over 0–240 min. The analytical method was validated; it showed high selectivity, linearity, and acceptable accuracy. We confirmed the lack of interaction of this cocktail in the control state (no effect of CYP inducer or inhibitor) and suggested AUC_ratio (metabolite/substrate) as a unit to evaluate DDI in vivo. In addition, the cocktail assay was applied for the determination of pharmacokinetic parameters against phenobarbital as a selective CYP2B inducer and ketoconazole as a strong CYP3A inhibitor. The concentration of cocktail substrates and the LC–MS/MS method were optimized. In conclusion, we developed a simultaneous and comprehensive analysis system for predicting potential DDI in mice.     

LC–MS/MS assay for olanzapine in human plasma and its application to a bioequivalence study

This paper describes a selective and sensitive assay for the determination of olanzapine (OLZ) in human plasma based on liquid chromatography–tandem mass spectrometry (LC–MS/MS). The analyte and quetiapine as internal standard (IS) were extracted from 200 μL plasma via solid phase extraction on Waters Oasis HLB cartridges. Chromatographic separation was achieved on an ACE 5C18-300 column (100 mm×4.6 mm, 5 μm) under isocratic conditions in a run time of 3.5 min. Mass spectrometric detection involved electrospray ionization in the positive ion mode followed by multiple reaction monitoring (MRM) of the transitions at m/z 313/256 for OLZ and m/z 384/253 for the IS. The assay was linear in the range 0.10–40.0 ng/mL with a lower limit of quantitation and limit of detection of 0.10 and 0.012 ng/mL, respectively. Intra- and inter-day precision (as coefficient of variation) and relative recovery were <5.0% and >90%, respectively. The method was successfully applied to a bioequivalence study of 5 and 10 mg OLZ disintegrating tablets in 40 healthy Indian males with reproducibility by incurred sample reanalysis in the range ?7.43 to 8.07%.     

Determination of 3α-hydroxytibolone by LC/MS/MS with electrospray ionization method and its application to bioequivalence study in human plasma

A specific and sensitive liquid chromatography?Ctandem mass spectrometry (LC/MS/MS) in the negative electrospray ionization mode was developed and validated in order to analyze 3??-hydroxytibolone (3??-OH-tibolone), major metabolite of tibolone in human plasma sample using only 200???L of plasma. 3??-OH-tibolone and 3??-OH-tibolone-d6 (internal standard, IS) in plasma were derivatized with p-toluenesulfonyl isocyanate after extraction using ethyl acetate. The dye residue and by-products was excluded using HLB Oasis SPE cartridge after derivertization. Separation of derivatized 3??-OH-tibolone and IS was performed on a C18 column within 6?min. The assay showed linearity in concentration ranges of 0.2?C20?ng/mL. Intra-day precision and accuracy ranged from 0.77 to 5.0?% and from 98.0 to 100.56?%, respectively. Inter-assay precision and accuracy ranged from 1.49 to 5.00?% and from 99.75 to 100.94?%, respectively. A specific, reproducible and sensitive LC/MS/MS method for quantification of 3??-OH-tibolone in human plasma was successfully applied to evaluate the bioequivalence of tibolone in human volunteer following single oral administration of 5?mg tibolone.     

Development and validation of a sensitive and selective UHPLC–MS/MS method for quantitation of an investigational anti-malarial compound, 2-tert-butylprimaquine (NP-96) in rat plasma,and its application in a preclinical pharmacokinetic study

An ultra-high performance liquid chromatographic tandem mass spectroscopy (UHPLC–MS/MS) method was developed and validated for the quantification of an investigational anti-malarial entity, 2-tert-butylprimaquine (NP-96), in rat plasma. Simple protein precipitation by acetonitrile was used for the sample preparation. Effective separation of NP-96, internal standard (IS) and matrix components were achieved on an UHPLC column (Hypersil Gold C18, 50 mm × 2.1 mm, 1.9 μm) using a mobile phase composed of acetonitrile and 20 mM ammonium acetate, which was pumped in a gradient mode at a flow rate of 450 μl/min. Selective reaction monitoring (SRM) was utilized for quantitation of the molecules. To increase sensitivity of the method, two ions of m/z 299 and m/z 231 were selected for NP-96, while IS was monitored for an ion of m/z 489. The method was validated according to FDA guideline on bioanalytical method validation and showed good compliance. The intra-day and inter-day precision expressed as R.S.D. was lower than 15% at all the tested quality control levels, including upper and lower limits of quantification. The calibration range was 2.5–500 ng/ml. Total runtime for the method was 5 min, which was suitable to produce high-throughput results for pharmacokinetic evaluation.