Stable-isotope dilution LC–ESI-MS/MS techniques for the quantification of total homocysteine in human plasma

Homocysteine is an endogenous sulphydryl aminoacid irreversibly catabolized by transsulfuration to cysteine or remethylated to methionine. Increased plasma levels of homocysteine are an independent risk factor for atherosclerosis and cardiovascular disease. Accurate and reliable quantification of this amino acid in plasma samples is essential in clinical practice to explore the presence of a hyperhomocysteinemia, for instance after an ischemic event, or to control a possible adjunctive risk factor in patients at higher risk. In this review, LC–ESI-MS/MS methods are discussed and compared with other analytical methods for plasma homocysteine. LC–ESI-MS/MS is a technique combining the physicochemical separation of liquid chromatography with the analysis of mass spectrometry. It is based on stable-isotope dilution and possesses inherent accuracy and precision. Quantitative analysis is achieved by using commercially available homocystine-d₈ as an internal standard. Taking advantage of the high sensitivity and specificity, approaches involving LC–ESI-MS/MS require less laborious sample preparation, no derivatization and produce reliable results.     

Simultaneous determination of a novel antifibrotic agent and three metabolites in human urine by LC–MS/MS

A robust and validated high performance liquid chromatography tandem mass spectrometry (LC–MS/MS) method has been developed for simultaneous determination of F351 (5-methyl-1-(4-hydroxylphenyl)-2-(1H)-pyridone) and three major metabolites in human urine sample. This assay method has also been validated in terms of selectivity, linearity, lower limit of quantification (LLOQ), accuracy, precision, stability, matrix effect and recovery. Chromatography was carried out on an XTerra RP 18 column and mass spectrometric analysis was performed using an API 4000 mass spectrometer coupled with electro-spray ionization (ESI) source in the positive ion mode. The MRM transitions of m/z 202 → 109, 232 → 93, 282 → 202 and 378 → 202 were used to quantify F351 and three metabolites, respectively. Retention times for F351 and three metabolites were 2.54, 1.38, 1.53 and 1.34 min, respectively. The assay was validated from 20 to 4000 ng/mL for F351 and M1, from 80 to16,000 ng/mL for M2 and M3. Intra- and inter-day precision for all analytes was <6.3%, method accuracy was between −11.2 and 0.3%. This assay was used to support a clinical study where multiple oral doses were administered to healthy subjects to investigate the pharmacokinetics, safety, and tolerability of F351.     

Simultaneous determination of citalopram and its metabolite in human plasma by LC–MS/MS applied to pharmacokinetic study

A simple sensitive and robust method for simultaneous determination of citalopram and desmethylcitalopram was developed using liquid chromatography tandem mass spectrometry (LC–MS/MS). A 200 μL aliquot of plasma sample was employed and deproteinized with methanol and desipramine was used as the internal standard. After vortex mixing and centrifugation, the supernatant was diluted with water (1:1, v/v) and then directly injected to analysis. Analytes were separated by a Zorbax XDB C₁₈ column with the mobile phase composed of acetonitrile and water (30:70, v/v) with 0.25% formic acid and monitored in MRM mode using a positive electrospray source with tandem mass spectrometry detection. The total run time was 3.5 min. The dynamic range was 0.2–100 ng/mL for citalopram and 0.25–50 ng/mL for desmethylcitalopram, respectively. Compared to the best existing literatures for plasma samples, the same LOQ for CIT (0.5 ng/mL) and lower LOQ for DCIT (0.25 vs 5 ng/mL) were reached, and less sample preparation steps and runtime (3.5 vs 10 min) were taken for our method. Accuracy and precision was lower than 8% and lower than 11.5% for either target. Validation results and its application to the analysis of plasma samples after oral administration of citalopram in healthy Chinese volunteers demonstrated the method was applicable to pharmacokinetic studies.     

Determination of metoclopramide in human plasma by LC–ESI-MS and its application to bioequivalance studies

An LC–MS method for the determination of metoclopramide in human plasma was developed and validated. Sample preparation involved extraction with ethyl acetate. Chromatographic separation was performed on a Thermo Hypersil-Hypurity C₁₈ (150 mm × 2.1 mm, 5 μm) with the mobile phase consisting of 40 mM ammonium acetate–methanol–acetonitrile. A single-quadrupole mass spectrometer with an electrospray interface was operated in the selected-ion monitoring mode to detect the [M+H]⁺ ions at m/z 300 for metoclopramide and at m/z 384 for the internal standard (prazosin). The method was validated over 0.78–50.00 ng mL^?1 for metoclopramide. The recovery was 67.8–83.1%, and the limit of quantitation (LOQ) detection was 0.78 ng mL^?1 for metoclopramide. The intra- and inter-day precision of the method at three concentrations was 5.0–13.6% with accuracy of 99.2–104.0%. Stability of compounds was established in a battery of stability studies. The method was successfully applied to bioequivalence studies of metoclopramide hydrochloride tablets to obtain the pharmacokinetic parameters.     

LC–MS/MS method development and validation for the determination of polymyxins and vancomycin in rat plasma

Simple, sensitive and robust liquid chromatography–tandem mass spectrometer (LC–MS/MS) methods were developed and validated for the determination of lipopeptide polymyxins and glycopeptide vancomycin in rat plasma. The effect of trichloroacetic acid (TCA) concentration on sample recoveries (peak area of sample recovered from plasma/peak area of sample from neat solvent solutions) was studied and an optimized concentration of 30% TCA were determined that gives the best sample recovery for the peptides from rat plasma. The effect of the TCA concentration on the chromatographic behavior of peptides was studied on a Phenomenex Jupiter C18 5 μ 300 Å 50 mm × 2 mm column using a mobile phase with a pH of 2.8. Other than protein precipitation, TCA also acted as ion pairing reagent and was only present in the samples but not in the mobile phases. The data demonstrated that by increasing the TCA concentration, the analyte retention and sensitivity were improved. The absence of TCA in mobile phase helped to reduce the ion source contamination and to achieve good reproducibility. The plasma method was linearly calibrated from 5 to 5000 ng/mL for polymyxins with precisions to be of 2.3–10.8%, and accuracies to be 91.7–107.4% for polymyxin B1, B2, E1, E2, respectively. For vancomycin the calibration is from 1 to 5000 ng/mL with precisions to be of 7.8–10.3 and accuracies to be 96.2–102.0%. The LLOQs corresponding with a coefficient of variation less than 20% were 7.5, 18.1, 7.3, 5.0 and 1.0 ng/mL for polymyxin B1, B2, E1, E2 and vancomycin, respectively.     

A simple and sensitive HPLC–ESI-MS/MS method for the determination of andrographolide in human plasma

A liquid chromatography–electrospray ionization tandem mass spectrometry (HPLC–ESI-MS/MS) method for the determination of andrographolide in human plasma was established. Dehydroandrographolide was used as the internal standard (I.S.). The plasma samples were deproteinized with methanol and separated on a Hanbon C₁₈ column with a mobile phase of methanol–water (70:30, v/v). HPLC–ESI-MS/MS was performed in the selected ion monitoring (SIM) mode using target ions at [M?H₂O–H]^?, m/z 331.1 for andrographolide and [M?H]^?, m/z 331.1 for the I.S. Calibration curve was linear over the range of 1.0–150.0 ng/mL. The chromatographic separation was achieved in less than 6.5 min. The lower limits of quantification (LLOQ) was 1.0 ng/mL. The intra and inter-run precisions were less than 6.95 and 7.22%, respectively. The method was successfully applied to determine the plasma concentrations of andrographolide in Chinese volunteers.     

Chromatographic separation of (E)- and (Z)-isomers of entacapone and their simultaneous quantitation in human plasma by LC–ESI-MS/MS

A selective, sensitive and high throughput liquid chromatography–tandem mass spectrometry (LC–ESI-MS/MS) method has been developed and validated for the chromatographic separation and quantitation of (E)-entacapone and (Z)-entacapone in human plasma. Sample clean-up involved liquid–liquid extraction (LLE) of both the isomers and carbamazepine used as internal standard from 500 μL of human plasma. Both the analytes were chromatographically separated with a resolution factor of 3.0 on a Gemini C18 (50 mm × 4.6 mm, 5 μm particle size) analytical column using 1% formic acid and methanol (50:50, v/v) as the mobile phase. The selectivity factor (α) of the column for the separation was 2.0, based on the capacity factors of 2.6 and 1.3 for (E)- and (Z)-isomers respectively. The parent → product ion transitions for both the isomers (m/z 306.1 → 233.0) and IS (m/z 237.3 → 194.2) were monitored on a triple quadrupole mass spectrometer, operating in the multiple reaction monitoring (MRM) and positive ion mode. The method was validated over the concentration range of 24.3–6076 ng/mL and 23.8–5960 ng/mL for (E)-entacapone and (Z)-entacapone respectively. Matrix effect was assessed by post-column analyte infusion experiment and the process/extraction efficiency found was 94.3% and 89.3% for (E)- and (Z)-isomers respectively. The method was successfully applied to a pivotal bioequivalence study in 36 healthy human subjects after oral administration of 200 mg (E)-entacapone tablet formulation under fasting conditions.     

Determination of free and glucuronidated kaempferol in rat plasma by LC–MS/MS: Application to pharmacokinetic study

Flavanoid kaempferol is mainly present as glucuronides and sulfates in rat plasma, and small amounts of the intact aglycone are also detected. In the this study, a rapid, specific and sensitive liquid chromatography–electrospray ionization-tandem mass spectrometry method (HPLC–MS/MS) was developed and validated for determination of kaempferol and its major metabolite glucuronidated kaempferol in rat plasma. A liquid–liquid extraction with acetic ether was involved for the extraction of kaempferol and internal standard. Analytes were separated on a C18 column (150 mm × 2.1 mm, 4.5 μm, Waters Corp.) with isocratic elution at a flow-rate of 0.3 ml min⁻¹. The mobile phase was consisted of 0.5% formic acid and acetonitrile (50:50, v/v). The Quattro Premier HPLC–MS/MS was operated under the multiple reaction-monitoring mode (MRM) using the electrospray ionization technique. The method was validated according to the FDA guidelines for validation of bioanalytical method. The validated method was successfully applied to the study of the pharmacokinetics in rats after oral administration of kaempferol with different doses.     

Determination of the unstable drug otilonium bromide in human plasma by LC–ESI-MS and its application to a pharmacokinetic study

Otilonium bromide (OB) degrades rapidly in plasma and readily undergoes hydrolysis by the plasma esterase. In this paper, an LC–ESI-MS method has been developed for the determination of OB in human plasma. The rapid degradation of OB in plasma was well prevented by immediate addition of potassium fluoride (KF, an inhibitor of plasma esterase) to the freshly collected plasma before prompt treatment with acetonitrile. The method was validated over the concentration range of 0.1–20 ng/ml. The data of intra-run and inter-run precision and accuracy were within ±15%. The mean extraction recoveries for OB and the internal standard were higher than 93.0% and the matrix effects were negligible. The method has been successfully used in a pharmacokinetic study.     

Quantification of isradipine in human plasma using LC–MS/MS for pharmacokinetic and bioequivalence study

A highly sensitive and rapid method for the analysis of isradipine in human plasma using liquid chromatography coupled to tandem mass spectrometry (LC–MS/MS) was developed. The procedure involves a simple liquid–liquid extraction of isradipine and amlodipine (IS, internal standard) with methyl-t-butyl ether after alkaline treatment and separation by RP-HPLC. Detection was performed by positive ion electrospray ionization (ESI) in multiple reaction monitoring (MRM) mode, monitoring the transitions m/z 372.1 → m/z 312.2 and m/z 408.8 → m/z 237.9, for quantification of isradipine and IS, respectively. The standard calibration curves showed good linearity within the range of 10 to 5000 pg/mL (r² ≥ 0.9998). The lower limit of quantitation (LLOQ) was 10 pg/mL. The retention times of isradipine (0.81 min) and IS (0.65 min) suggested the potential for high throughput of the proposed method. In addition, no significant metabolic compounds were found to interfere with the analysis. This method offered good precision and accuracy and was successfully applied for the pharmacokinetic and bioequivalence studies of 5 mg of sustained-release isradipine in 24 healthy Korean volunteers.     

Simultaneous determination of histamine and Nτ-methylhistamine in human plasma and urine by gas chromatography-mass spectrometry

A new and sensitive method is described for the determination of histamine and N^τ-methylhistamine in human plasma and urine by gas chromatography-mass spectrometry. ¹⁵N₂-Labeled histamine and N^τ-[methyl-d₃]methylhistamine were used as internal standards. Histamine and N^τ-methylhistamine were converted to the derivatives N^α-heptafluorobutyryl-N^τ-ethoxycarbonylhistamine and N^α-heptafluorobutyryl-N^τ-methylhistamine, respectively. After these derivatives had been purified on a small column packed with CPG-10, the molecular ions were monitored during selected ion monitoring. Linear standard curves were obtained in the range of 0.5–10 ng/ml for both compounds. The reliability of the histamine analysis was demonstrated by using two different ion pairs, while a comparison with results from two different derivatizations on the same urine sample also established the specificity of the N^τ-methylhistamine analysis. An increase of 1 ng of histamine in the plasma could be precisely determined by the present method. The histamine content of plasma from five normal subjects was determined as 0.83 ÷ 0.37 (S.D.) ng/ml and the N^τ-methylhistamine content in most subjects was below the limits of this measurement. High excretion of histamine was noted in the urine collected in the early morning from a patient with nephritis.     

Improved method for the determination of cyclic guanosine monophosphate (cGMP) in human plasma by LC–MS/MS

Cyclic guanosine monophosphate (cGMP) is an important second messenger molecule involved in gating ion channels and activating protein kinases. Here, we describe a validated LC–MS/MS method for the quantification of cGMP in human plasma, utilizing a stable isotope labeled analogue of cGMP as I.S. Plasma samples were extracted and concentrated by weak anion exchange solid phase extraction and the extracts were chromatographically separated on a porous graphitic carbon column. The analytes were detected by positive electrospray ionization and tandem mass spectrometry. The calibration function was linear in the range 1–20 nM and the intra- and inter-day precision showed relative standard deviations of better than 2 and 6%, respectively. The accuracy was always better than 4%. Plasma concentrations in healthy human subjects determined with this method were 3.92 ± 1.17 nM (n = 20). The method was, due to its isotope labeled I.S., matrix independent.     

LC–MS/MS method using unbonded silica column and aqueous/methanol mobile phase for the simultaneous quantification of a drug candidate and co-administered metformin in rat plasma

BMS-754807 and metformin were co-administered in drug discovery studies which required the quantitation of both compounds in plasma. Since the two compounds are chemically and structurally dissimilar, developing a single bioanalytical method presented a number of chromatographic challenges including the achievement of appropriate retention times and peak shapes on a single analytical column. To address this chromatographic challenge, we investigated different LC columns under different gradient elution schemes using aqueous/organic mobile phases. Using unbonded silica column and aqueous/methanol mobile phase, we were able to obtain robust and well-resolving chromatographic conditions to support the development and implementation of a single LC–MS/MS bioanalytical method. The use of sub-2 micron particle sizes and a high flow rate, which are attainable with UPLC systems, enhanced the method. The method performance evaluation showed that the method easily met the normally used acceptance criteria for bioanalytical methods, namely a deviation of ±15% from the nominal concentration except at lower limit of quantitation (LLOQ), where ±20% is accepted. The reported LLOQ of 7.8 ng/ml, for both BMS-754807 and metformin, was adequate to support the pharmacokinetic studies.     

Simultaneous determination of tolbutamide,omeprazole, midazolam and dextromethorphan in human plasma by LC–MS/MS—A high throughput approach to evaluate drug–drug interactions

Drug–drug interactions involving cytochrome P450 (CYP450s) are an important factor for evaluation of a new chemical entity (NCE) in drug development. To evaluate the potential inhibitory effects of a NCE on the pharmacokinetics of a cocktail of representative probes of CYP enzymes (midazolam for CYP3A4, tolbutamide for CYP2C9, omeprazole for CYP2C19 and dextromethorphan for CYP2D6) and the safety and tolerability of the NCE in the presence of probe substrates, a high throughput liquid chromatography/tandem mass spectrometry (LC–MS/MS) method was developed and validated for the simultaneous determination of tolbutamide, omeprazole, midazolam and dextromethorphan in human plasma using tolbutamide-d₉, midazolam-d₄, (±)-omeprazole-d₃, and dextromethorphan-d₃ as the internal standards (ISs). Human plasma samples of 50 μL were extracted by a simple protein-precipitation procedure and analyzed using a high performance liquid chromatography electrospray tandem mass spectrometer system. Reversed-phase HPLC separation was achieved with a Hypersil GOLD AQ column (50 mm × 4.6 mm, 5 μm). MS/MS detection was set at mass transitions of 271 → 172 m/z for tolbutamide, 346 → 198 m/z for omeprazole, 326 → 291 m/z for midazolam, 272 → 171 m/z for dextromethorphan, 280 → 172 m/z for tolbutamide-d₉ (IS), 349 → 198 m/z for (±)-omeprazole-d₃ (IS), 330 → 295 m/z for midazolam-d₄ (IS), and 275 → 171 m/z for dextromethorphan-d₃ (IS) in positive mode. The high throughput LC–MS/MS method was validated for accuracy, precision, sensitivity, stability, recovery, matrix effects, and calibration range. Acceptable intra-run and inter-run assay precision (<10%) and accuracy (<10%) were achieved over a linear range of 50–50,000 ng/mL for tolbutamide, 1–1000 ng/mL for omeprazole, 0.1–100 ng/mL for midazolam and 0.05–50 ng/mL for dextromethorphan in human plasma. Method robustness was demonstrated by the 100% pass rate of 24 incurred sample analysis runs and all of the 50 clinical study samples used for incurred sample reproducibility (ISR) test having met the acceptance criterion (%Diff within 20%). The overall ISR results for all compounds showed that over 95% of the samples had a %Diff of less than 10%. The method is simple, rapid and rugged, and has been applied successfully to sample analysis in support of a drug–drug interaction study.     

Development and validation of a sensitive and rapid non-aqueous LC–ESI-MS/MS method for measurement of diosgenin in the plasma of normal and hyperlipidemic rats: A comparative study

A sensitive and specific electrospray ionization liquid chromatography–tandem mass spectrometry method was developed to detect diosgenin in the plasma of normal and hyperlipidemic rats. Diosgenin was extracted with n-hexane–ethyl acetate (9:1, v/v) using sarsasapogenin as an internal standard. With multiple reaction monitoring modes, linear calibration curves were obtained in the range 10–1500 ng/mL (r ≥ 0.9979) and the limit of quantification was 10 ng/mL. Intra- and inter-assay variabilities were within 7.74%, and accuracies were between ?5.33% and 1.50%. The assay was successfully applied to study pharmacokinetics in rats after oral administration of diosgenin. Significantly different pharmacokinetics between normal and hyperlipidemic rats were observed, which would be beneficial for the clinical use of diosgenin.     

Simultaneous determination of cyclophosphamide and carboxyethylphosphoramide mustard in human plasma using online extraction and electrospray tandem mass spectrometry (HTLC–ESI-MS/MS)

A rapid and selective method for simultaneous determination of cyclophosphamide and its metabolite carboxyethylphosphoramide mustard (CEPM) was developed using online sample preparation and separation with tandem mass spectrometric detection. Diluted plasma was injected onto an extraction column (Cyclone MAX 0.5 mm × 50 mm, >30 μm), the sample matrix was washed with an aqueous solution, and retained analytes were transferred to an analytical column (Gemini 3 μm C18 110A, 100 mm × 2.0 mm) using a gradient mobile phase prior to detection by MS/MS. Analytes were detected in an API-3000 LC-MS/MS system using positive multiple-reaction monitoring mode (m/z 261/140 and 293/221 for CTX and CEPM, respectively). Online extraction recoveries were 76% and 72% for cyclophosphamide and CEPM. Within-day and between-day variabilities were <3.0%, and accuracies were between ?6.9% and 5.2%. This method has been used to measure plasma cyclophosphamide and CEPM concentrations in an ongoing Phase II study in children with newly diagnosed medulloblastoma.     

Investigation of an on-line two-dimensional chromatographic approach for peptide analysis in plasma by LC–MS–MS

Reversed phase and hydrophilic interaction chromatography (HILIC) were successfully coupled for the on-line extraction and quantitative analysis of peptides by ESI–LC–MS/MS. A total of 11 peptides were utilized to determine the conditions for proper focusing and separation on both dimensions. Minor modifications to the initial organic composition of the first reversed-phase dimension provided options between a comprehensive (generic) or more selective approach for peptide transfer to the second HILIC dimension. Ion-pairing with trifluoroacetic acid (TFA) provided adequate chromatographic retention and peak symmetry for the selected peptides on both C₁₈ and HILIC. The resulting signal suppression from TFA was partially recovered by a post-column “TFA fix” using acetic acid yielding improvements in sensitivity. Minimal sample preparation aligned with standard on-line extraction procedures provided highly reproducible and robust results for over 300 sequential matrix injections. Final optimized conditions were successfully employed for the quantitation of peptide PTHrP (1–36) in rat K₃EDTA plasma from 25.0 to 10,000 ng/mL using PTHrP (1–34) as the analog internal standard. This highly orthogonal two-dimensional configuration was found to provide the unique selectivity required to overcome issues with interfering endogenous components and minimize electrospray ionization effects in biological samples.     

Rapid and sensitive LC–MS/MS assay for the quantitation of 20(S)-protopanaxadiol in human plasma

This paper describes a rapid and sensitive method for the quantitation of 20(S)-protopanaxadiol (PPD) in human plasma based on high-performance liquid chromatography–tandem mass spectrometry (LC–MS/MS). The analyte and internal standard (I.S.), ginsenoside Rh₂, were extracted from plasma by liquid–liquid extraction and separated on a Zorbax extend C₁₈ analytical column using methanol–acetonitrile-10 mM ammonium acetate (47.5:47.5:5, v/v/v) as mobile phase. Detection was by tandem mass spectrometry using electrospray ionization in the positive ion mode and multiple reaction monitoring (MRM). The assay was linear over the concentration range 0.1–100.0 ng/ml with a limit of detection of 0.05 ng/ml. The method was successfully applied to a clinical pharmacokinetic study in healthy volunteers after a single oral administration of a PPD 25 mg capsule.