Levels Of Quality Control Samples Research Articles

ANALYTICAL METHOD DEVELOPMENT AND VALIDATION FOR ESTIMATION OF EZETIMIBE IN RABBIT PLASMA USING REVERSED-PHASE HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY

Objectives: The objective of the present work was to develop an analytical method and validation for the estimation of Ezetimibe in rabbit plasma using high-performance liquid chromatography (HPLC). Methods: A simple, rapid, sensitive, and accurate HPLC method was developed and validated for the quantification of Ezetimibe concentration in rabbit plasma using metoclopramide as an internal standard. Separation was performed on the Xerra C18 column (250 × 4.6 mm 5 μm) using a mobile phase consisting of 0.1% perchloric acid: acetonitrile(55:45 v/v) at a flow rate of 1 ml/min. Validation of the method was performed to demonstrate its selectivity, linearity, precision, accuracy, ruggedness, recovery, and matrix effect. Results: The calibration curves of Ezetimibe were linear over a concentration range of 5–1022 μg/mL. The with-in and between-day coefficients of variation were <10%. The extraction recoveries of Ezetimibe at the three levels of quality control samples were 99.961%, 99.767%, and 99.938%. Conclusion: The method was rapid with a retention time of Ezetimibe and the internal standard observed at 10 min, respectively. The developed method was successfully applied to studying the pharmacokinetics of Ezetimibe in rabbits.

Accurate determination of Biotinidase activity in serum by HPLC and its utilization as second tier test for the confirmation of initial positive newborn screening results

Diagnosis of Biotinidase deficiency (BTD) is extremely important to avoid several neurodevelopmental problems in early childhood. Colorimetric and fluorometric methods lack specificity and selectivity due to several interferences resulting in a high number of false positive results. We developed an HPLC method for BTD activity in serum with fluorescent detection. In colorimetric assays, biotinidase attacks the amide linkage of the artificial substrate biotinidyl-4-aminobenzoic acid (B-PABA) and releases p-aminobenzoic acid (PABA), which is converted to a purple dye by diazotization reaction. The newly developed method injects the reaction mixture directly into the HPLC column and quantifies using a six-point calibration curve without coupling and diazotization reaction. The method is linear over the 5–1000 μmol/L range. The detection and quantitation limits were 2.5 μmol/L and 5.0 μmol/L, respectively. When compared with colorimetric assay, the correlation coefficient (R2) was 0.9963. The within-assay and between-assay precision was <10.0% for four levels of quality control samples. No significant variation in BTD activity was detected due to hemolysis, icteric, and lipemic samples. The newly developed method eliminates the potential interference due to the presence of aromatic amines and significantly reduces the false positive results observed with the colorimetric method. It is simple, specific, sensitive, faster in sample preparation, and requires a small sample volume. The newly developed HPLC method was used in our laboratory as a secondary tier test for initial positive BTD samples from newborn screening programs. To our knowledge, no similar HPLC method has been reported to date.

Quantitation of meropenem in dried blood spots using microfluidic-based volumetric sampling coupled with LC-MS/MS bioanalysis in preterm neonates

Meropenem, a carbapenem antibiotic, has been used for empirical and definitive therapy of severe infections for many years. Therapeutic drug monitoring (TDM) plays an indispensable role in the individualization of meropenem particularly in the preterm neonates, a population in which adjusting proper dosages has always been one of the most challenging tasks for their growth changes. In this report, a simple and accurate method for the quantitative analysis of meropenem in dried blood spot (DBS) samples by LC-MS/MS was developed. The traditional DBS drawbacks were conquered in this study by combining microfluidic-based volumetric sampling, shorten drying procedure, and sensitive detection. Moreover, the on-card stability of meropenem was improved obviously. The DBS-based method validation included hematocrit (Hct) effect, selectivity, carry-over, linearity, accuracy, precision, matrix effect, recovery and stability (high temperature and humidity). The calibration linear range of meropenem was 0.3–100 µg/mL. The acceptance criteria of accuracy (relative error < 4.53 %) and precision (coefficient of variation < 8.63 %) were met in all levels of quality control samples. The DBS samples was stable at 40 °C for 12 h, room temperature for 1 day, 4 °C for 7 days, −20 °C for 14 days and −40 °C for 30 days, respectively. A good correlation was observed between DBS concentration and plasma concentration of meropenem. There was 93.4 % of the samples between estimated plasma concentration and plasma concentration within 20 % of the mean of concentration, and no significant Hct effect was observed on the quantification. It has been successfully applied to samples derived from preterm neonates with severe infections. The supported data indicated that the DBS-based method using microfluidic-based volumetric sampling could be an alternative strategy to carry on TDM of meropenem in preterm neonates, with satisfactory performance and logistics advantages.

BIOM-25. DETERMINATION OF LEVELS OF SMALL MOLECULAR WEIGHT THIOLS AND DISULFIDES IN MOUSE AND HUMAN NORMAL BRAIN AND GLIOBLASTOMA TISSUE

Abstract BACKGROUND Small molecular weight (SMW) thiols play an essential role in many biochemical processes, particularly in maintaining redox homeostasis. Herein, we report on the development and qualification of a sensitive and rapid LC-MS/MS method for the determination of oxidative stress biomarkers glutathione (GSH), cysteine (Cys), glutathione disulfide (GSSG), cystine (CySS), as well as the interrelated precursors and products methionine (Met), homocysteine (Hcy), cystathionine (Cysth), gamma glutamyl-cysteine (Glu-Cys), and cysteinylglycine (Cys-Gly) in both mouse and human normal brain and glioblastoma tissue. METHODS Mouse and human tissue samples were homogenized with N-ethylmaleimide solution to prevent thiol oxidation. Analytes were then extracted from homogenate samples by protein precipitation with 2% formic acid in acetonitrile. Separate calibration curves were prepared for thiols and disulfides in analytical solutions using stable isotope-labeled internal standards (IS). Three levels of quality control samples were prepared in mouse and human brain homogenates. The detection was performed on Sciex QTRAP 6500+ mass spectrometer in positive electrospray ionization mode. RESULTS The concentration ranges of 0.04-40 µmol/L for Glu-Cys/Hcy, 4-400 µmol/L for Met/GSH/Cys/Cys-Gly and 0.05-20 µmol/L for GSSG/CySS/Cysth were established using a linear regression model. A gradient elution was applied on Intrada Amino Acid column to achieve the optimal chromatographic separation with a 5 minute total run time. At least three precision and accuracy runs were conducted on different days. The stability of analytes in biological matrix and in analytical solutions has been tested at room temperature, 4 °C, and -80 °C for appropriate sample preparation, analysis, and storage. CONCLUSIONS A bioanalytical method to quantify a panel of SMW thiols and disulfides has been successfully developed, qualified, and utilized to assess the levels of these biomolecules in mouse normal brain and glioblastoma tissue as well as in glioblastoma tissue of patients enrolled in the sonodynamic therapy clinical trial (NCT04559685).

UPLC-MS/MS assay for the simultaneous determination of catecholamines and their metabolites at low pg/mg in rat/mouse striatum

Catecholamines and their metabolites act as neurotransmitters in the brain and are important for nervous system function. In the current work, a highly selective and sensitive UPLC-MS/MS assay was developed for quantitation of six catecholamines and their metabolites, including dopamine, norepinephrine, serotonin, 3,4-dihydroxyphenylacetic acid, homovanillic acid and 5-hydroxyindolacetic acid from rat and mouse striatum as pharmacodynamic biomarkers to support neuroscience and pharmaceutical research. A fit-for-purpose strategy for method development, assay qualification and study support were adopted for this assay. A surrogate matrix (brain homogenizing solution absent of targeted analytes) was used for preparation of calibration samples and certain levels of quality control samples to avoid interference from endogenous baselines. Homogenized rodent striatum was derivatized by dansyl chloride to enhance the sensitivity, followed by liquid-liquid extraction with ethyl acetate in 96-well plate format. The lower limit of quantitation (LLOQ) was 0.2 ng/mL in tissue homogenate, equivalent to 3.2 pg/mg in brain tissue, which could be further reduced to ten times lower by changing the re-dissolving and injecting volume in the last sample purification step. Acceptable accuracy, precision, linearity, specificity, recovery, and matrix effect was obtained. Bench-top stability (2 h), freeze-thaw stability (3 cycles at −20 °C), and − 80 °C storage stability (up to 51 days) in both tissue homogenate and surrogate matrix along with autosampler stability (60 h at 4°C) all met acceptance criteria. This assay was successfully applied to measure the six analytes in striatum from mice treated with the neurotoxin 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), an animal model of Parkinsonism for which dosing protocols can vary widely, and further confirmed the metabolic pathway of neurotoxicity by the quantification of catecholamine metabolites. Our study is the first detailed the step-by-step recovery and pointed out the key factors for the assay to simultaneously quantify these six neurotransmitters in rodent striatum with superior sensitivity.

Absolute quantification of senescence mediators in cells using multiple reaction monitoring liquid chromatography-Tandem mass spectrometry

BackgroundThe identification of unique senescence markers remains challenging. Current hallmarks of senescent cells, including increased senescence-associated β-galactosidase activity, increased levels of cell cycle regulators such as p16INK4a, p27, and p53, and altered levels of sirtuins and lamins, are detected commonly by Western blot and immunohistochemistry methods. Mass spectrometry outperforms these conventional quantification methods in terms of high throughput, specificity, and reproducibility. ObjectivesTo develop multiple reaction monitoring-based tandem mass spectrometric senescence assay for simultaneous measuring of p16INK4a, p27, p53, p53-β, the seven proteins of the sirtuins family and the four transcript variants of lamins proteins in aging cell model and cancerous cell lines. MethodologyMultiple reaction monitoring-tandem mass transitions per protein were developed for each signature peptide(s) and stable isotope-labeled internal standard. The developed assay was validated in a matrix using breast cancer MCF7 cell lines according to the US-FDA guidelines for bioanalytical assays. ResultsThe analytes chromatographic peaks were baseline separated and showed linear behavior in a wide dynamic range with r2 ≥ 0.98. The method for all proteins has passed the inter/intra-day precision and accuracy validation using three levels of quality control samples. The accuracy and the precision for most analytes were 80–120% and ≤20%, respectively. The method's sensitivity for the panels' signature peptides ranged from 1 ng μL−1 to 1 μg mL−1. Extraction recovery assessed in two quality control levels was >60% for most analytes. This LC-MS-MS validated senescence assay showed reduced lamin A, lamin A△10, lamin A△50, SIRT1, SIRT3, SIRT5, p53, and p16INK4a, as well as p53-β induction, are implicated in replicative senescence. Meanwhile, increased lamin C: lamin A ratio was evident and can diagnose breast carcinogenesis. Moreover, in breast cancer metastasis, reduced SIRT2 and p27 and elevated levels of lamin A△50, SIRT5, SIRT7, and p53-β are evident. ConclusionLC-MS/MS is a potent alternative tool to the currently available assays. The high throughput method established can study senescence's role in different pathophysiological processes.

Development and validation of an LC-MS/MS method for quantifying nine antimicrobials in human serum and its application to study the exposure of Chinese pregnant women to antimicrobials.

BackgroundTo study the prevalence of the exposure of pregnant women to antimicrobials, a sensitive and reliable liquid chromatography‐tandem mass spectrometry (LC‐MS/MS) method was developed and validated to determine nine antimicrobials, namely sulfadimidine, sulfapyridine, sulfadiazine, sulfathiazole, ofloxacin, ciprofloxacin, norfloxacin, tetracycline, and lincomycin, in human serum.MethodsThe sample preparation procedure included protein precipitation followed by a cleanup step with solid phase extraction (SPE). Separation was carried out using a CORTECS T3 column (100 × 2.1 mm, 2.7 µm) by gradient elution with a runtime of 8.0 min. Detection was performed on a triple quadruple tandem mass spectrometer with scheduled multiple reaction monitoring (sMRM) in positive ion scan mode.ResultsThe calibration curves were linear over the concentration range of 0.5–50 ng/ml, and the limit of quantitation was between 0.01 and 0.2 ng/ml. For each level of quality control samples, the inter‐ and intra‐assay precision values were less than 12.0%, and the accuracy ranged from 86.1% to 109.0%. No significant matrix effect or carryover was observed. The antimicrobials of interest were stable under all investigated conditions. The validated method was applied to analyze clinical samples from pregnant women in China, and 10 out of 500 samples showed the presence of antimicrobial residues. Moreover, compared with the time‐resolved fluoro‐immunoassay (TRFIA) method, the developed method showed greater sensitivity and specificity.ConclusionThis study provides a simple and rapid LC‐MS/MS method for the simultaneous measurement of nine antimicrobials in serum samples, which could be a useful tool in clinical utilization.

An adapted LC-MS/MS method for the determination of free plasma concentration of cefoperazone in children: Age-dependent protein binding

Antimicrobial activity of cefoperazone, a high protein bound cephalosporin, depends on its unbound concentration. However, the protein binding data of cefoperazone in children is limited, making it challenging to optimize antimicrobial therapy in pediatric clinical practice. Furthermore, a validated method to measure the free part in children is unavailable with the small volume of samples that can be obtained. Therefore, in the present study, we developed and validated an LC-MS/MS method for the determination of free cefoperazone in children. In this study, 70 μL of plasma was used to prepare the ultrafiltrate (only containing the free drug). Chromatographic separation of the analyte was achieved on a C18 column using gradient elution with a mobile phase of acetonitrile and water (0.1% formic acid). Negative electrospray ionisation in the multiple reaction monitoring mode was applied for the detection of cefoperazone and ceftiofur (internal standard). The calibration curve was prepared in the range of 5–5000 ng/mL with excellent linearity. For each level of quality control samples, the intra- and inter-day precision (CV) was below 9.0%, and the accuracy ranged from 91.5% to 105.0%. The matrix effect was less than 11.7%, and the recovery was between 92.9% and 95.9% of cefoperazone. The validated method has been successfully applied to the determination of free plasma concentration of cefoperazone in pediatric patients. The results of the unbound fraction showed considerable individual variability (range: 8.1–48.0%). The correlation analysis showed that age and albumin had significant effects on the protein binding of cefoperazone.

LC-MS/MS quantitative analysis of phylloquinone, menaquinone-4 and menaquinone-7 in the human serum of a healthy population.

A novel application of the liquid chromatography method combined with the triple quadrupole tandem mass spectrometry method was developed for the quantification of vitamin K1 and two forms of vitamin K2 (menaquinone-4, menaquinone-7) in human serum. Total chromatography time for each run was 9 min. Time required for the sample pretreatment procedures was approximately 4 h. The coefficients of variation (CVs) of intra-assay were 10.4%, 3.2 % and 2.3% for vitamin K1 in three levels of quality control samples; were 14.3%, 3.2% and 6.7% for menaquinone-4; and were 11.1%, 6.0% and 7.0% for menaquinone-7. The inter-assay CVs were 12.8%, 11.3% and 7.4% for vitamin K1; were 15.2%, 9.2% and 8.7% for menaquinone-4; and were 13.2%,11.1% and 7.2% for menaquinone-7. No interference was found between K1, menaquinone-4 and menaquinone-7, nor any deuterated internal standards. This method was then used to determine reference values for Caucasian populations of central European origin. Samples were measured from 191 healthy volunteers (51.2 ± 16.2 years (mean ± SD)) and the values concerning K1 were 0.044–1.357 ng/mL for women and 0.030–1.214 ng/mL for men. The values for menaquinone-4 and menaquinone-7 did not exhibit any differences between women and men, and were 0.050–1.598 and 0.074–0.759 ng/mL, respectively.

Development and Validation of a Bioanalytical method for the Determination of Levamisole Residue in Backyard Poultry Egg

Development of a bioanalytical method is often tricky and labour intensive due to its nature or the criteria set by regulatory bodies. In the present study, a simple, reliable and sensitive analytical method was developed for the determination of levamisole residue in chicken eggs. Levamisole and ronidazole (internal standard, IS) were separated on a C18 reversed-phase HPLC column. Following liquid-liquid extraction, chromatographic separation was accomplished with a mobile phase consisting of acetonitrile: 0.05M KH2PO4 at ratio of 20: 80, and the drug was detected at 235nm using a UV detector at a flow rate of 1.0ml/min at the ambient temperature. Linearity was obtained over the range 0.3125–10.0μg/ml for levamisole hydrochloride with lower limit of quantitation of 0.3125μg/ml. For each level of quality control samples, inter-and intra-day precision (%CV) was within ±15%. Absolutes extraction recovery of drug from plasma was ≥;60%. The residue limit of levamisole gradually increased from day 1 to day 3 and then declined from day4 to day 7. The highest level of residue after oral administration of 80mg/kg levamisole to chickens was on day 3 (0.599μg/g). This level was lower than the recommended maximum residue limit (MRL), thus, no withholding period was required for egg consumption following the administration of the levamisole solution.

A simple HPLC-UV method for the quantification of theophylline in rabbit plasma and its pharmacokinetic application.

A simple, precise and accurate high-performance liquid chromatography-ultraviolet method was developed and validated for the quantification of theophylline in rabbit plasma using hydroxyethyl theophylline as an internal standard. Separation was performed on Waters(®) C18 column (µBondapak™ 5 µm, 150 × 3.9 mm) using a mobile phase consisting of water-acetonitrile (96:4 v/v) at a flow rate of 1 mL/min. Validation of the method was performed in order to demonstrate its selectivity, linearity, precision, accuracy and stability. The calibration curves of theophylline were linear over a concentration range of 0.1-25 µg/mL. The within- and between-day coefficient of variation (CV) were <10%. The extraction recoveries of theophylline at the three levels of quality control samples were 63.1, 69.4 and 69.7%. The method was rapid with retention time of theophylline and the internal standard observed at ∼5.2 and 6.5 min, respectively. The developed method was applied successfully for studying the pharmacokinetics of theophylline in rabbits.

Quantitative hydrophilic interaction chromatography-mass spectrometry analysis of N-acetylneuraminic acid and N-acetylmannosamine in human plasma.

N-acetylneuraminic acid (Neu5Ac or NANA) is the most predominant sialic acid in mammals. As a terminal component in many glycoproteins and glycolipids, sialic acid is believed to be an important biomarker related to various diseases. Its precursor, N-acetylmannosamine (ManNAc), is being investigated as a potential treatment for GNE myopathy. In this work, we developed two highly sensitive and selective liquid chromatography-tandem mass spectrometry (LC-MS/MS) methods for the quantitation of ManNAc and free Neu5Ac in human plasma. A fit-for-purpose approach was adopted during method validation and sample analysis. To measure the endogenous compounds and overcome the interference from plasma samples, a surrogate matrix that contained 5% bovine serum albumin (BSA) was used for the preparation of calibration standards and certain levels of quality control (QC) samples. QC samples at higher concentrations were prepared in the authentic matrix (human plasma) to best mimic incurred samples. For both methods, an Ostro 96-well phospholipid removal plate was used for sample extraction, which efficiently removed the phospholipids from the plasma samples prior to LC injection, eliminated matrix effect, and improved sensitivity. Chromatographic separation was achieved using hydrophilic interaction chromatography (HILIC) and gradient elution in order to retain the two polar compounds. The lower limit of quantitation (LLOQ) for ManNAc and Neu5Ac was 10.0 and 25.0ng/mL, respectively. The overall accuracy of the two assays was within 100%±8.3% based on three levels of QC samples. Inter- and intra-run precision (coefficient of variation (%CV)) across three analytical runs was less than 6.7% for ManNAc and less than 10.8% for Neu5Ac. These methods have been validated to support clinical studies.

A Simple HPLC Bioanalytical Method for the Determination of Doxorubicin Hydrochloride in Rat Plasma: Application to Pharmacokinetic Studies

Purpose: To develop a simple, accurate, and precise high performance chromatography (HPLC) method with spectrophotometric detection for the determination of doxorubicin hydrochloride in rat plasma. Methods: Doxorubicin hydrochloride and daunorubicin hydrochloride (internal standard, IS) were separated on a C18 reversed-phase HPLC column. Following protein precipitation extraction, chromatographic separation was accomplished with a mobile phase consisting of acetonitrile: water at ratio of 30:70 (pH 3.0), and the drug was detected at 233 nm using a UV detector at flow rate of 1.0 ml/min and ambient temperature. Results: Linearity was obtained over the range 1.0 – 50.0 μg/ml for doxorubicin hydrochloride with lower limit of quantitation of 1.0 μg/ml. For each level of quality control samples, inter- and intra-day precision (% CV) was < 9.6 and 5.1 %, respectively. Stability of doxorubicin hydrochloride in plasma was within the acceptance limit (± 15 %) with no evidence of degradation during sample processing and 30 days storage in a deep freezer at -70 ± 5 °C. Absolutes extraction recovery of drug from plasma was ≥ 86 %. Conclusion: The method is highly selective and rugged for the determination of doxorubicin hydrochloride in rat plasma and should be suitable for conducting pharmacokinetic studies and therapeutic drug monitoring.

Improved ruggedness of an ion‐pairing liquid chromatography/tandem mass spectrometry assay for the quantitative analysis of the triphosphate metabolite of a nucleoside reverse transcriptase inhibitor in peripheral blood mononuclear cells

Nucleotide analogs are highly polar and ionic, which impose great challenges on bioanalysis. Ion-pairing liquid chromatography/tandem mass spectrometry (LC/MS/MS) is the predominant reported approach for such compounds. Assay ruggedness of ion-pairing LC/MS/MS methods was often a challenge due to the potential contamination of the ion source of the mass spectrometer and LC column performance deterioration caused by ion-pairing reagents. An ion-pairing reagent was only added to the reconstitution solution to minimize its exposure to the MS ion source. To achieve optimum sensitivity, high pH mobile phases and negative ion ESI were needed for the LC/MS/MS method. However, high pH mobile phases led to the accumulation of ion-pairing reagent on the analytical column, which was washed off with an acidic solution to restore the column performance. In addition, isopropanol was used as a mobile phase modifier to improve peak shape and sensitivity. The limit of detection was established at 1.0 ng/mL in the cell lysate. The calibration curve showed good linearity over the range of 1.0 to 100 ng/mL. The overall accuracy was no less than 87.7% based on four levels of quality control samples. Inter-run precision and intra-run precision across four analytical runs for low, geometric, medium and high QCs were less than 12.9. By identifying and addressing the root cause of the assay ruggedness problem, we have developed a rugged ion-pairing LC/MS/MS method for a triphosphate (TP) metabolite of BMS-986001 in peripheral blood mononuclear cells. The new method overcame challenges such as a rapid deterioration of the peak shape, increased carryover and extremely poor column life. The peak shape was well maintained throughout multiple analytical runs. This method has been successfully applied to a toxicology study in cynomolgus monkey.

An HPLC–ESI-MS method for simultaneous determination of fourteen metabolites of promethazine and caffeine and its application to pharmacokinetic study of the combination therapy against motion sickness

The combination therapy, promethazine and caffeine had been proven effective in treating motion sickness and counteracting some possible side effects of using promethazine alone while the mechanism and interaction remained unclear. Therefore, an HPLC–ESI-MS method for simultaneous determination of both drugs, and their metabolites was developed for purpose of pharmacokinetic study. To determine as many metabolites as possible, the influence of parameters such as column, flow rate and pH value of mobile phase, ionization polarity and fragmentation voltage were optimized. Fourteen target analytes were well separated and all of them could be identified and determined in plasma after administration of promethazine and caffeine. The LODs and LOQs were 0.9–6.0 and 2.50–16.0ng/ml, respectively; the recoveries of three levels of quality control samples were from 86.7% to 102%; the intra-day and inter-day precisions were less than 3% and 9%, separately; and the RSDs of compound stability were all lower than 10% within 24h after sample preparation. As a pharmacokinetic study of the combination therapy in 30 healthy volunteers, concentration–time curves of the drugs and metabolites were studied. The present method for simultaneous measurement of more than ten metabolites is valuable for the study of mechanism and interaction of the combination therapy.

Development of a multiplex UPLC-MRM MS method for quantification of human membrane transport proteins OATP1B1, OATP1B3 and OATP2B1 in in vitro systems and tissues

OATP1B1, OATP1B3 and OATP2B1 are important members of the organic anion transporting polypeptides (OATP) family and are implicated in the hepatic disposition of endobiotics and xenobiotics. Quantitating the expression levels of human OATP1B1, OATP1B3 and OATP2B1 in in vitro systems and tissue samples could significantly improve attempts to scale up in vitro data and result in more effective in vitro–in vivo correlation of transporter-mediated effects on drug disposition, such as hepatic clearance. In the present study, a quantification method was developed, characterized, and implemented for simultaneous determination of human OATP1B1, OATP1B3 and OATP2B1 in HEK cells transfected with OATP-expressing plasmid vectors (SLCO1B1, SLCO1B3, and SLCO2B1, respectively), human hepatocytes, human brain capillary endothelial cells, and humanized mouse liver tissue using UPLC-MRM MS. Purified membrane protein standards prepared and characterized as previously reported (Protein Expr. Purif. 2008, 57, 163-71) were first used as standards for absolute quantification of the expression levels of the three human OATP membrane proteins. The specificity of the optimized MRM transitions were characterized by analyzing the tryptic digests of the membrane protein fraction of wild type HEK cells and control mouse liver tissue using the herein reported UPLC-MRM MS method. The linearity of the calibration curve spanned from 0.2μgmL−1 (0.040μgmg−1) to 20μgmL−1 (4.0μgmg−1), with accuracy (% RE) within 15% at all concentrations examined for all three OATPs of interest in this study. The intra- and inter-day assay accuracy (% RE) and coefficient of variations (% CV) of triplicates are all within 15% for all levels of quality control samples prepared by mixing the membrane fraction of control mouse liver tissue with the required amount of purified human OATP1B1, OATP1B3 and OATP2B1.

Simultaneous determination of seven flavonoids in dog plasma by ultra-performance liquid chromatography–tandem mass spectrometry and its application to a bioequivalence study of bioactive components in Herba Epimedii and Er-Xian Decoction

In this study, a sensitive and specific ultra-performance liquid chromatography–tandem mass method has been developed and validated for the identification and determination of 7 flavonoids in dog plasma for the first time: epimedin A, epimedin B, epimedin C, icariin, sagittatoside B, 2″- O-rhamnosyl icariside II, and baohuoside I. Chromatographic separation was accomplished on an Agilent Zorbax-SB C 18 column (50 mm × 2.1 mm, 1.8 μm) with a gradient elution system composed of 0.3% acetic acid and 0.3% acetic acid in acetonitrile at a flow rate of 0.4 mL/min. Detection was based on a triple quadrupole mass spectrometer using a multiple reaction monitoring mode with an electrospray ionization source. All of the calibration curves showed good linearity ( r > 0.99) within the tested concentration ranges. The lower limits of quantification of the seven analytes were all lower than 0.0654 ng/mL. The relative standard deviations for intra- and inter-batches of the seven analytes were less than 13.7% and 14.9%, respectively, at four concentration levels of quality control samples, and the recoveries were between 92.8% and 114.5%, respectively. In addition, the seven flavonoids were found to be stable in dog plasma samples under short- and long-term storage and processing conditions. The validated method was successfully applied to a bioequivalence study in dog plasma after the oral administration of extracts of Herba Epimedii and Er-Xian Decoction.

Development and validation of a rapid and sensitive assay for simultaneous quantification of midazolam, 1′-hydroxymidazolam, and 4-hydroxymidazolam by liquid chromatography coupled to tandem mass-spectrometry

Midazolam is an ultra short acting benzodiazepine derivative and a specific probe for phenotyping cytochrome P450 (P450) 3A4/5 activity. A rapid, sensitive, and selective LC–MS/MS method was developed for simultaneous quantitation of midazolam and its metabolites (1′-hydroxymidazolam and 4-hydroxymidazolam). Deuterated (D 5) analog of midazolam was utilized as an internal standard. Sample preparation either from human plasma (100 μL) or liver microsomal incubations involved a simple protein precipitation using acetonitrile (900 μL) with an average recovery of >90% for all compounds. The chromatographic separation was achieved using Zorbax-SB Phenyl, Rapid Resolution HT (2.1 mm × 100 mm, 3.5 μm) and a gradient elution with 10 mM ammonium acetate in 10% methanol (A) and acetonitrile (B). The flow rate was 0.25 mL/min and total run time was 5.5 min. Calibration curves were linear over the concentration range of 0.100–250 ng/mL. The lower limit of quantitation (LLOQ) was 0.1 ng/mL for all three analytes. The accuracy and precision, estimated at LLOQ and three concentration levels of quality control samples in six replicates, were within 85–115%. In conclusion, a robust, simple and highly sensitive analytical method was developed and validated for the analysis of midazolam and its metabolites. This method is suitable for characterizing the P450 3A4/5 activity in vitro or in human pharmacokinetic studies allowing administration of smaller doses of midazolam.

A simple and rapid high‐performance liquid chromatography method for determination of alendronate sodium in beagle dog plasma with application to preclinical pharmacokinetic study

A simple and rapid high performance liquid chromatographic (HPLC) method for quantifying alendronate in beagle dog plasma was developed, validated and applied to a pharmacokinetic study. The sample preparation involved coprecipitation with CaCl(2) and derivatization with o-phthalaldehyde. Chromatographic separation was achieved on a Diamonsil C(18 )(250 x 4.6 mm, 5 microm) using acetonitrile-0.4% EDTA-Na(2) (16:84, v/v) containing 0.034% of NaOH as mobile phase. The fluorimetric detector was operated at 339 nm (excitation) and 447 nm (emission). The linearity over the concentration range of 5.00-600 ng/mL for alendronate was obtained and the lower limit of quantification was 5.00 ng/mL. For each level of quality control samples, inter-day and intra-day precisions were less than 8.52 and 7.42% and accuracies were less than 9.07%. The assay was applied to the analysis of samples from a pharmacokinetic study. Following the oral administration of 70 mg alendronate sodium to beagle dogs, the maximum plasma concentration (C(max)) and elimination half-life were 152 +/- 27.3 and 1.75 +/- 0.267 h, respectively. The method was demonstrated to be highly feasible and reproducible for pharmacokinetic studies.

A rapid and specific approach for direct measurement of donepezil concentration in human plasma by LC‐MS/MS employing solid‐phase extraction

A selective, rapid and simple liquid chromatography-tandem mass spectrometry (LC-MS/MS) method is described for assay of donepezil in human plasma using escitalopram as an internal standard. Chromatographic separation was achieved on a Betabasic-C(8), 5 microm, 100 x 4.6 mm column using methanol:water:formic acid (90:9.97:0.03, v/v/v) as mobile phase. Detection of donepezil and internal standard was achieved by ESI MS/MS in positive ion mode using 380.20/91.10 and 325.13/262.00 transitions, respectively. The linearity over the concentration range of 0.15-50 ng/mL for donepezil was obtained and the lower limit of quantification was 0.15 ng/mL. For each level of quality control samples, inter-day and intra-day precisions (RSD) were < or =8.92 and 10.35% and accuracy (%RE) were < or =7.33% and 9.33%, respectively. The recovery was more than 88.50% for both donepezil and internal standard by solid-phase extraction, eliminating evaporation and reconstitution steps.