Accurate determination of drug-to-antibody ratio of interchain cysteine-linked antibody-drug conjugates by LC-HRMS.

Accurate determination of the drug-to-antibody ratio (DAR) of interchain cysteine-linked antibody-drug conjugates (ADCs) is challenging. High-resolution mass spectrometry (HRMS) analysis of the ADCs at the intact or subunit level provides a feasible way to measure the DAR. However, the measured DAR is usually lower than the true DAR because of the variation in ionization efficiency between different DAR species. In this work, we developed a novel standard-free HRMS method involving isotope-labeled payload conjugation, protease digestion, and liquid chromatography-HRMS (LC-HRMS) analysis for accurate determination of the DAR of the interchain cysteine-linked ADCs with cleavable or non-cleavable linkers. Isotope-labeled payload conjugations eliminated the structural and chemical differences between different DAR species and ensured that the drugs or payload-containing peptides could be separated from each other in the mass spectrometer. A papain digestion strategy for ADCs with cleavable linkers showed a DAR of 3.79, with a relative standard deviation (RSD) of 0.48 (n = 3). Similarly, the trypsin and chymotrypsin digestion strategy that is applicable to ADCs with non-cleavable linkers showed a DAR of 3.77 and an RSD of 0.86 (n = 3). The DAR determined by this method was consistent with the DAR of the ADCs that was measured by the UV/Vis method. This method will be very useful to researchers working in the field of ADC discovery and development. Graphical abstract.

Characterization of Positional Isomers of Interchain Cysteine Linked Antibody-Drug Conjugates by High-Resolution Mass Spectrometry.

Interchain cysteine linked antibody-drug conjugates (ADCs) are emerging therapeutic products that antagonize cancers. The toxic payloads are selectively linked to the interchain cysteines and generate heterogeneous mixtures of positional isomers. These positional isomers might contribute differently to the therapeutic efficacy because of the variation in conjugation stability, and thus they need to be well characterized. However, the characterization of the positional isomers of interchain cysteine linked ADCs is very challenging, mainly because of the high similarity between those isomers. In this research, we developed a novel mass spectrometry method for the characterization of positional isomers of interchain cysteine linked ADCs. The subunit analysis and the bottom-up analysis provided abundant information about the drug numbers and drug linking positions on each chain. Because the method can provide accurate data on drug linking numbers and positions on each chain, it will be very useful for researchers in cancer drug development and cancer treatment.

Development and validation of an LC-MS/MS Method for the quantitation of heparan sulfate in human urine.

Heparan sulfate is a linear polysaccharide and serves as an important biomarker to monitor patient response to therapies for MPS III disorder. It is challenging to analyze heparan sulfate intact owing to its complexity and heterogeneity. Therefore, a sensitive, robust and validated LC-MS/MS method is needed to support the clinical studies for the quantitation of heparan sulfate in biofluids under regulated settings. Presented in this work are the results of the development and validation of an LC-MS/MS method for the quantitation of heparan sulfate in human urine using selected high-abundant disaccharides as surrogates. During sample processing, a combination of analytical technologies have been employed, including rapid digestion, filtration, solid-phase extraction and chemical derivatization. The validated method is highly sensitive and is able to analyze heparan sulfate in urine samples from healthy donors. Disaccharide constitution analysis in urine samples from 25 healthy donors was performed using the assay and demonstrated the proof of concept of using selected disaccharides as a surrogate for validation and quantitation.

Quantitation of P450 3A4 endogenous biomarker - 4ß-hydroxycholesterol - in human plasma using LC/ESI-MS/MS.

4ß-Hydroxycholesterol (4ß-HC) has been proposed as a new endogenous biomarker for cytochrome P450 3A4/5 activity. Therefore, it is important to have a robust method for its accurate determination in human plasma. Here a liquid chromatography-tandem mass spectrometry with electrospray ionization (LC/ESI-MS/MS) assay for the quantitation of 4ß-HC in human plasma is described. While the calibration standards were prepared in a surrogate matrix for human plasma, the quality control samples were prepared in human plasma to mimic the incurred study samples. In order to achieve accurate determination of 4ß-HC, the chromatographic separation of 4ß-HC from its isomers, especially 4α-hydroxycholesterol (4α-HC), was crucial. In the absence of an authentic 4α-HC standard at the time of this study, an alternative selectivity test strategy was developed to confirm the separation. After being alkalized with potassium hydroxide, the human plasma sample (50 µL) was extracted with hexane, derivatized into picolinyl esters using picolinic acid, extracted again with hexane, and then analyzed by LC/ESI-MS/MS. The calibration curve range was 5-500 ng/mL and the chromatographic separation was achieved on a 50 × 2.1 mm Thermal Hypersil Gold column with a gradient elution. The assay accuracy, precision, linearity, selectivity and analyte stability throughout the analysis were established. The validated assay was successfully applied to a Phase I clinical study for the measurement of 4ß-HC in human plasma.

Pilot and pivotal study to evaluate the bioequivalence of two paroxetine 40 mg tablet formulations in healthy Chinese subjects.

The purpose of this study was to conduct a pilot study in order to obtain reliable results for further planning of a well-designed pivotal trial comparing the bioequivalence (BE) of two paroxetine tablet formulations in healthy Chinese subjects. Before conducting the pivotal trial, the pilot trial enrolled 14 subjects to help in study design, establishing the recruitment period, determining pharmacokinetics (PK) time points and sample size, and assessing BE of the two formulations. The single-center, randomized, open-label, single-dose, two period crossover study with a 7-day washout interval was conducted after obtaining information from the fasted pilot trial in 72 healthy volunteers for a pivotal study under fed and fasted conditions, respectively. There were 19 PK sample collection time points employed in both the pilot and pivotal trials. A sensitive and specific liquid chromatography- tandem mass spectrometry (LC-MS/ MS) method was developed and validated for determining paroxetine in human plasma. BE between two articles was determined by calculating 90% confidence intervals (CIs) for the ratio of Cmax 91.38 - 110.39% for the pilot trial, 99.81 - 114.08% for pivotal trial under fasted condition, and 94.06 - 110.41% for pivotal trial under fed condition, AUC(0-t) 96.06 - 110.52% for pilot study, 100.88 - 113.05% for the pivotal trial under fasted condition, and 97.08 - 106.06% for pivotal study under fed condition, and AUC(0-∞) 96.17 - 110.42% for the pilot study, 100.85 - 112.81% for the pivotal trial under fasted condition and 97.22 - 106.14% for the pivotal study under fed condition, respectively. These values for the test and reference products are within the 80 - 125% interval proposed by FDA and EMEA. It was concluded that the proposed method was successfully applied to a PK study in healthy Chinese volunteers, and results showed from both the pilot and pivotal studies that the two paroxetine formulations are bioequivalent in their rates and extent of absorption.

Determination of metformin in rat plasma by HILIC-MS/MS combined with Tecan automation and direct injection.

Metformin is a well-known oral antihyperglycemic drug used in treatment of type II diabetes. Analysis of metformin in biological fluids is a challenge owing to its high polarity and small molecular size, which lead to poor retention of metformin on reversed-phase liquid chromatographic columns. A high-throughput method was developed and validated for the determination of metformin in rat plasma in support of preclinical toxicology studies, using hydrophilic interaction liquid chromatography tandem mass spectrometry (HILIC-MS/MS) and Tecan automated sample preparation. Extracted samples were directly injected onto the unbounded silica column with an aqueous-organic mobile phase. This HILIC-MS/MS method was validated for accuracy, precision, sensitivity, stability, matrix effect, recovery and calibration range. Acceptable intra-run and inter-run assay precision (coefficient of variation ≤ 3.9%) and accuracy (99.0-101.8%) were achieved over a linear range of 50-50,000 ng/mL. Metformin is stable in rat plasma for at least 6 h at room temperature, 147 days at -70°C and through three freeze (-70°C) and thaw cycles. Metformin is also stable in rat whole blood for at least 2 h at room temperature and in an ice-water bath. The validated method was successfully used in support of several preclinical studies where metformin is dosed together with an investigational drug substance. The ruggedness of the validated method was demonstrated by the incurred sample reproducibility test.

Development and validation of an LC-MS/MS method for the quantification of fascin proteins in human serum.

Background: Fascin is an actin-bundling protein that has been linked to tumor cell migration, invasion, metastasis, disease progression and mortality, thus serving as a novel cancer biomarker. Bioanalytical methods to measure fascin in biological matrices are sparsely reported, while accurate quantitation of fascin levels may lend support for fascin as a promising therapeutic target. Method: An LC-MS/MS-based method involving protein precipitation, enzymatic digestion and solid phase extraction was developed and validated for the quantitation of fascin in human serum. Linearity over a calibration range of 5-500 ng/ml with a LLOQ of 5 ng/ml, great accuracy and precision, excellent parallelism as well as high extraction recovery were achieved. Conclusion: This method provides a valuable tool for anticancer drug development and cancer treatment.

Simultaneous quantification of total antibody and antibody-conjugated drug for XMT-1522 in human plasma using immunocapture-liquid chromatography/mass spectrometry.

XMT-1522, an antibody-drug conjugate (ADC) currently in Phase I clinical development, represents the first Dolaflexin®-based, cleavable ADC with a high drug-antibody ratio (DAR). In this work, a novel immunocapture LC-MS/MS method was successfully developed for the simultaneous quantification of both total antibody and cleavable antibody-conjugated drug auristatin F-hydroxypropylamide (AF-HPA) in human plasma. This method utilized microwave-assisted enzymatic digestion for the total antibody and chemical release of the drug from ADC on a 96-well based immunocapture sample preparation platform. The total antibody and the conjugated drug AF-HPA were separated and subsequently quantified concurrently by LC-MS/MS. The linear range of the standard curve for total antibody was from 50 to 5000 ng/mL and for AF-HPA was from 3.3 to 330 ng/mL. The linearities showed R2 ≥ 0.993 for total antibody and R2 ≥ 0.996 for AF-HPA, respectively. The intra- and inter-day precision and accuracy were well within 15%. The validated method, with the characteristics of high efficiency, great selectivity, free of carryover, short LC-MS/MS time (˜3.5 min) and low sample volume (20 µl), was successfully applied for analyzing Phase 1 cancer patient samples.

Development and validation of LC-MS/MS methods for the quantification of the novel anticancer agent guadecitabine and its active metabolite ß­decitabine in human plasma, whole blood and urine.

Guadecitabine (SGI-110), a dinucleotide of ߭decitabine and deoxyguanosine, is currently being evaluated in phase II/III clinical trials for the treatment of hematological malignancies and solid tumors. This article describes the development and validation of bioanalytical assays to quantify guadecitabine and its active metabolite ߭decitabine in human plasma, whole blood and urine using high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS). Since ߭decitabine is rapidly metabolized further by cytidine deaminase, plasma and whole blood samples were kept on ice-water after collection and stabilized with tetrahydrouridine (THU) directly upon sample collection. Sample preparation consisted of protein precipitation for plasma and whole blood and dilution for urine samples and was further optimized for each matrix and analyte separately. Final extracts were injected onto a C6-phenyl column for guadecitabine analysis, or a Nova-Pak Silica column for ߭decitabine analysis. Gradient elution was applied for both analytes using the same eluents for each assay and detection was performed on triple quadrupole mass spectrometers operating in the positive ion mode (Sciex QTRAP 5500 and QTRAP 6500). The assay for guadecitabine was linear over a range of 1.0-200 ng/mL (plasma, whole blood) and 10-2000 ng/mL (urine). For ߭decitabine the assay was linear over a range of 0.5-100 ng/mL (plasma, whole blood) and 5-1000 ng/mL (urine). The presented methods were successfully validated according to the latest FDA and EMA guidelines for bioanalytical method validation and applied in a guadecitabine clinical mass balance trial in patients with advanced cancer.

Application of LC-MS for quantitative analysis and metabolite identification of therapeutic oligonucleotides.

Therapeutic oligonucleotides (OGNs) have been studied extensively in the recent years as novel agents designed to selectively and specifically inhibit target gene expression in cell culture, in animal disease models and in human. This review summarizes applications of liquid chromatography coupled with mass spectrometry or tandem mass spectrometry (LC-MS or LC-MS/MS) for quantitative analysis of therapeutic OGNs and characterization of their metabolism in vitro and in vivo described in the literature over the past 10 years. Although the applications of LC-MS or LC-MS/MS to the molecular mass measurement, sequence determination, DNA adducts identification, detection of mutations and characterization of covalent and/or noncovalent DNA/RNA complexes have been comprehensively reviewed in a few excellent review papers. The quantitative bioanalysis and metabolite identification of therapeutic OGNs using LC-MS or LC-MS/MS have not been covered. This review covers technical issues, current approaches and applications of LC-MS or LC-MS/MS for quantitative analysis of OGNs in biological matrices and characterization of their in vitro and in vivo metabolism. Finally, some conclusions are drawn and prospects of LC-MS in quantitative analysis and metabolism characterization of therapeutic OGNs are discussed.

ICP-MS determination of serum aluminum in monkeys subcutaneously administered an alhydrogel-formulated drug candidate.

AIM: To develop and validate an inductively coupled plasma-mass spectrometry method for quantitative bioanalysis of aluminum (Al) in monkey serum in support of a GLP TOX study with alhydrogel-formulated drug candidate. METHODS & RESULTS: The method was linear over a dynamic range of 10-1000 ng/ml using a 50-µl sample volume. The intra-/inter-run precision (%CV) of the quality control sample results were ≤7.9% (CV) and the accuracy (%bias) within ±11.0% across all quality control concentrations evaluated. Other validation parameters, including stability under various conditions, extraction recovery and matrix effect, all met the acceptance criteria. CONCLUSION: The validated method was successfully implemented for the quantitative analysis of Al in monkey serum to assess the systemic exposure to Al.

An ultra-sensitive LC-MS/MS method to determine midazolam levels in human plasma: development, validation and application to a clinical study.

AIM: Midazolam is a commonly used marker substrate for the in vivo assessment of CYP3A activity. Reliable pharmacokinetic assessment at sub-pharmacological doses of midazolam requires an ultra-sensitive analytical method. METHODS: A new, ultra-sensitive LC-MS/MS method for the determination of midazolam in human plasma using SPE was developed and fully validated. The lowest limit of quantitation is 0.1 pg/ml with a sample volume of 500 µl. RESULTS/CONCLUSION: The following parameters were validated: sensitivity, assay accuracy and precision, linearity, selectivity, and stability of midazolam at pertinent analytical and storage conditions. The validated method was utilized successfully for the sample assay during a midazolam microdosing study for the evaluation of CYP3A4 activity of a clinical candidate.

The 10th GCC Closed Forum: rejected data, GCP in bioanalysis, extract stability, BAV, processed batch acceptance, matrix stability, critical reagents, ELN and data integrity and counteracting fraud.

The 10th Global CRO Council (GCC) Closed Forum was held in Orlando, FL, USA on 18 April 2016. In attendance were decision makers from international CRO member companies offering bioanalytical services. The objective of this meeting was for GCC members to meet and discuss scientific and regulatory issues specific to bioanalysis. The issues discussed at this closed forum included reporting data from failed method validation runs, GCP for clinical sample bioanalysis, extracted sample stability, biomarker assay validation, processed batch acceptance criteria, electronic laboratory notebooks and data integrity, Health Canada's Notice regarding replicates in matrix stability evaluations, critical reagents and regulatory approaches to counteract fraud. In order to obtain the pharma perspectives on some of these topics, the first joint CRO-Pharma Scientific Interchange Meeting was held on 12 November 2016, in Denver, Colorado, USA. The five topics discussed at this Interchange meeting were reporting data from failed method validation runs, GCP for clinical sample bioanalysis, extracted sample stability, processed batch acceptance criteria and electronic laboratory notebooks and data integrity. The conclusions from the discussions of these topics at both meetings are included in this report.

UPLC-MS/MS assay for the simultaneous determination of ethinyl estradiol, norgestimate and 17-Desacetyl norgestimate at low pg/mL in human plasma.

Previously, because of the difficulty of measuring very low levels (pg/mL) of norgestimate (NGM) due to rapid metabolism to its active and major metabolite, 17-Desacetyl norgestimate (DNGM), only DNGM and the co-administered ethinyl estradiol (EE2) were required to be analyzed in bioequivalence (BE) studies for oral contraceptive NGM/EE2 tablets. Recently, with more sensitive assays available, health authorities have requested that bioequivalence of NGM be also demonstrated in addition to DNGM and EE2. Therefore, it was important to establish a 3-in-1 method for the quantitation of EE2, NGM and DNGM in human plasma. Here a UPLC-MS/MS method for the simultaneous quantitation of EE2, NGM and DNGM in human plasma at low pg/mL range is described. EE2, NGM, DNGM and their isotopic labeled internal standards (IS) EE2-d4, NGM-d6 and DNGM-d6 in 0.4mL of human plasma were extracted with hexane/ethyl acetate. The extracts were evaporated to dryness and derivatized with dansyl chloride, to enhance the mass spec response. The derivatives were reconstituted with methanol and analyzed by UPLC-MS/MS. In order to minimize the ex-vivo conversion of NGM to DNGM, sodium fluoride/potassium oxalate was used as anti-coagulant. To achieve desirable throughput for sample analysis, UPLC-MS/MS with a run time of 4.4min was utilized. The calibration curve ranges were 5-500pg/mL for EE2 and NGM, and 25-2500pg/mL for DNGM, respectively. The chromatographic separation was achieved on a Waters Acquity UPLC HSS T3 (100×2.1mm, 1.8µm) column with a gradient elution. Assay accuracy, precision, linearity, selectivity, sensitivity and analyte stability covering sample storage and analysis were established. This validated UPLC-MS/MS method has been applied to a BE study for the determination of EE2, NGM and DNGM concentrations in human plasma.

9th GCC closed forum: CAPA in regulated bioanalysis; method robustness, biosimilars, preclinical method validation, endogenous biomarkers, whole blood stability, regulatory audit experiences and electronic laboratory notebooks.

The 9th GCCClosed Forum was held just prior to the 2015 Workshop on Recent Issues in Bioanalysis (WRIB) in Miami, FL, USA on 13 April 2015. In attendance were 58 senior-level participants, from eight countries, representing 38 CRO companies offering bioanalytical services. The objective of this meeting was for CRO bioanalytical representatives to meet and discuss scientific and regulatory issues specific to bioanalysis. The issues selected at this year's closed forum include CAPA, biosimilars, preclinical method validation, endogenous biomarkers, whole blood stability, and ELNs. A summary of the industry's best practices and the conclusions from the discussion of these topics is included in this meeting report.

Simultaneous determination of glycyrrhizin, a marker component in radix Glycyrrhizae, and its major metabolite glycyrrhetic acid in human plasma by LC-MS/MS.

Glycyrrhizin (GLY) which has been widely used in traditional Chinese medicinal preparation possesses various pharmacological effects. In order to investigate the pharmacokinetic behavior of GLY in human after oral administration of GLY or licorice root, a liquid chromatography/tandem mass spectrometry (LC-MS/MS) method was developed and validated for the simultaneous determination of GLY and its major metabolite glycyrrhetic acid (GA) in human plasma. The method involved a solid phase extraction of GLY, GA, and alpha-hederin, the internal standard (IS), from plasma with Waters Oasis MCX solid phase extraction (SPE) cartridges (30 mg) and a detection using a Micromass Quattro LC liquid chromatography/tandem mass spectrometry system with electrospray ionization source in positive ion mode. Separation of the analytes was achieved within 5min on a SepaxHP CN analytical column with a mobile phase of acetonitrile:water (50:50, v:v) containing 0.1% formic acid and 5mM ammonium acetate. Multiple reaction monitoring (MRM) was utilized for the detection monitoring 823--> 453 for GLY, 471--> 177 for GA and 752--> 456 for IS. The LC-MS/MS method was validated for specificity, sensitivity, accuracy, precision, and calibration function. The assay had a calibration range from 10 to 10,000 ng/mL and a lower limit of quantification of 10 ng/mL for both GLY and GA when 0.2 mL plasma was used for extraction. The percent coefficient of variation for accuracy and precision (inter-run and intra-run) for this method was less than 11.0% with a %Nominal ranging from 87.6 to 106.4% for GLY and 93.7 to 107.8% for GA. Stability of the analytes over sample processing (freeze/thaw, bench-top and long-term storage) and in the extracted samples was also tested and established.

In vitro plasma protein binding determination of flunarizine using equilibrium dialysis and liquid chromatography-tandem mass spectrometry.

A highly sensitive method was developed and validated for determining the free fraction of flunarizine in human plasma. Equilibrium dialysis was used for the separation of free (unbound) drug and liquid chromatography/tandem mass spectrometry (LC-MS/MS) was used for quantitation. Post-dialysis plasma or buffer samples of 0.2 mL were extracted using a liquid-liquid extraction procedure and analyzed using a high performance liquid chromatography electrospray tandem mass spectrometer system. The compounds were eluted isocratically on a Supelco Supelcosil ABZ+Plus column, ionized using a positive ion atmospheric pressure electrospray ionization source, and analyzed using multiple reaction monitoring. The ion transitions monitored were m/z 405-->203 for flunarizine and m/z 409-->207 for flunarizine-d4 (internal standard, IS). The chromatographic run time was 3.5 min per injection, with retention times of 2.1 min for both flunarizine and IS. The calibration curve for flunarizine was linear over the concentration range of 0.25-2000 ng/mL (r(2)>0.9989) in the combined matrix of human plasma and isotonic sodium phosphate buffer (1:1, v/v) with the lower limit of quantitation of 0.25 ng/mL. The inter-assay coefficient of variability (CV) for the quality control samples was less than 13.5%, and the inter-assay percent nominal was greater than 98.2%. In vitro protein binding of flunarizine was determined at concentrations of 5, 10 and 100 microg/mL using the validated method. Flunarizine was extensively bound to plasma protein with a 0.083+/-0.005% overall percent free drug in plasma and a CV value less than 7.8%. This validated method will be used for the ex vivo assessment of flunarizine protein binding in human plasma from a drug-drug interaction clinical study.

Simultaneous determination of glipizide and rosiglitazone unbound drug concentrations in plasma by equilibrium dialysis and liquid chromatography-tandem mass spectrometry.

Glipizide and rosiglitazone are widely used to treat Type 2 diabetes. In order to investigate drug-drug protein binding interaction between glipizide and rosiglitazone, a method was developed and validated for simultaneously determining the free (unbound) fraction of glipizide and rosiglitazone in plasma employing equilibrium dialysis for the separation of free drug and liquid chromatography-tandem mass spectrometry (LC-MS/MS) for quantitation. Post-dialysis human plasma or buffer samples of 0.2 ml were extracted using a liquid-liquid extraction procedure and analyzed by a high performance liquid chromatography electrospray tandem mass spectrometer system. The compounds were eluted isocratically on a Zorbax SB-Phenyl column, ionized using an atmospheric pressure electrospray ionization source and analyzed in positive ion mode with multiple reaction monitoring. The ion transitions monitored were m/z 446-->321 for glipizide, m/z 358-->135 for rosiglitazone, and m/z 271-->155 for tolbutamide (internal standard, IS). The chromatographic run time was 5 min per injection, with retention times of 2.3, 3.4 and 2.3 min for glipizide, rosiglitazone and IS, respectively. The calibration curves of glipizide and rosiglitazone were over the range of 1-2000 ng/ml (r(2)>0.9969) in the combined matrix of human plasma and isotonic sodium phosphate buffer (1:1, v/v). The inter-assay precision and accuracy of the quality control samples were <10.9% of coefficient of variability and >93.5% and 94.5% of nominal concentration for glipizide and rosiglitazone, respectively. The lower limit of quantitation of both glipizide and rosiglitazone was 1.0 ng/ml. Both glipizide and rosiglitazone bound to plasma protein extensively (>99% bound). Glipizide and rosiglitazone free fraction averaged 0.678+/-0.071 and 0.389+/-0.061%, respectively, at plasma concentration of 1000 ng/ml. This developed method proves reproducible and sensitive and its application to clinical samples is also reported.

Development and validation of a sensitive liquid chromatographic-tandem mass spectrometric method for the determination of cromolyn sodium in human plasma.

Cromolyn sodium is a safe compound with potent anti-allergic properties when used locally or topically. Clinical data from systemic exposure is not available because of the poor GI absorption when given orally. In order to evaluate a new approach to enhance the absorption and bioavailability of cromolyn sodium, a sensitive assay was needed to support an oral-dose study in humans. This paper describes a liquid chromatographic-tandem mass spectrometric (LC-MS-MS) method for the analysis of cromolyn sodium in human plasma. The method consists of a two-step extraction with subsequent analysis using a high-performance liquid chromatography electrospray tandem mass spectrometer system. The compounds were eluted isocratically on a C(18) column followed by a backflush. The total run time is 6 min. The standard curve of cromolyn sodium was over the range of 0.313 to 750 ng/mL with a lower limit of quantitation (LLOQ) of 0.313 ng/mL when 0.5 mL of plasma was used for analysis. The percent coefficient of variation (C.V.) for accuracy and precision (inter-assay and intra-assay) was less than 15% over the validated concentration range and the coefficients of determination, r(2), were >0.991577. The method is simple, sensitive, and selective, and has been successfully utilized for oral cromolyn sodium clinical studies.

Simultaneous determination of pioglitazone and its two active metabolites in human plasma by LC-MS/MS.

A liquid chromatography/tandem mass spectrometry (LC-MS/MS) method was developed and validated for the simultaneous determination of pioglitazone (PIO) and its two metabolites: M-III (keto-derivative) and M-IV (hydroxy-derivative) in human plasma. Human plasma samples of 0.2 ml were extracted by a single step liquid-liquid extraction procedure and analyzed using a high performance liquid chromatography (HPLC) electrospray tandem mass spectrometer system. The compounds were eluted isocratically on a C-18 column, ionized using a positive ion atmospheric pressure electrospray ionization source and analyzed using multiple reaction monitoring mode. The ion transitions monitored were m/z 357-->134 for PIO, m/z 371-->148 for M-III, m/z 373-->150 for M-IV and m/z 413-->178 for the internal standard. The chromatographic run time was 2.5 min per injection, with retention times of 1.45, 1.02 and 0.95 min for PIO, M-III and M-IV, respectively. The calibration curves of pioglitazone, M-III and M-IV were well fit over the range of 0.5-2000 ng/ml (r(2)>0.998759) by using a weighted (1/x(2)) quadratic regression. The inter-day precisions of the quality control samples (QCs) were