LC–MS/MS method with chemical derivatization for quantitation of L‐threonate in human plasma

An LC–MS/MS‐based bioanalytical method has been developed to measure the concentration of L‐threonate at its endogenous level in human plasma. Following isotope dilution and protein precipitation, the samples were acetylated and chromatographed under reversed‐phase conditions for baseline separation of the derivatized L‐threonate and its stereoisomer D‐erythronate. The method was assessed by a fit‐for‐purpose validation with a calibration range from 100 to 10,000 ng/mL. The intra‐run coefficients of variation (CVs) were <3.6% and the inter‐run CV was 3.2% for the QC samples at endogenous level. At the lower limit of quantitation, the intra‐run CV was 6.1% and the average inaccuracy was ?1.4%. This method provides an efficient and reliable quantitation of L‐threonate and could be useful to certain biomarker investigators.     

Determination of pethidine in human plasma by LC–MS/MS

A sensitive and selective liquid chromatography–tandem mass spectrometry method for the determination of pethidine in human plasma was developed and validated over the concentration range of 4–2000 ng/mL. After addition of ketamine as internal standard, liquid–liquid extraction was used to produce a protein‐free extract. Chromatographic separation was achieved on a 100 × 2.1 mm, 5 µm particle, Allure^TM PFP propyl column, with 45:40:15 (v/v/v) acetonitrile–methanol–water containing 0.2% formic acid as mobile phase. The MS data acquisition was accomplished by multiple reactions monitoring mode with positive electrospray ionization interface. The lower limit of quantification was 4 ng/mL; for inter‐day and intra‐day tests, the precision (RSD) for the entire validation was less than 7%, and the accuracy was within 95.9–106.5%. The method is sensitive and simple, and was successfully applied to analysis of samples of clinical intoxication. Copyright © 2010 John Wiley & Sons, Ltd.     

Development and validation of an LC–MS/MS quantitative method for endogenous deoxynucleoside triphosphates in cellular lysate

The endogenous deoxynucleoside triphosphate (dNTP) pool includes deoxyadenosine triphosphate (dATP), deoxycytidine triphosphate (dCTP), deoxyguanosine triphosphate (dGTP) and thymidine triphosphate (TTP). The endogenous dNTP pool is regulated by complex enzymatic pathways that can be targeted by drugs, such as antimetabolites. In addition, these components compete with antiviral nucleos(t)ide analog triphosphates, contributing to the mechanism of pharmacologic action. This communication describes the development and validation of a sensitive method to quantify the intracellular dNTP pool in cellular lysates. The extraction process was optimized for dNTPs using an indirect strategy – the separation of mono‐, di‐ and triphosphate moieties by strong anion exchange, dephosphorylation of target fractions to molar equivalent nucleosides – followed by sensitive quantitation using liquid chromatography–tandem mass spectrometry. The validated analytical range was 50–2500 fmol/sample for each dNTP. The assay was used to quantify dNTPs in peripheral blood mononuclear cells from 40 clinical research participants (n = 279 samples). Median (interquartile range) concentrations were 143 (116, 169) for dATP, 737 (605, 887) for dCTP, 237 (200, 290) for dGTP and 315 (220, 456) for TTP, in femtomoles per million cells. This method allows for future studies of endogenous dNTP disposition in subjects receiving antimetabolites or nucleos(t)ide analogs, or for other clinical scenarios.     

Analytical validation and clinical application of urinary vanillylmandelic acid and homovanillic acid by LC–MS/MS for diagnosis of neuroblastoma

Vanillylmandelic acid (VMA) and homovanillic acid (HVA) are clinical biomarkers for diagnosis of neuroblastoma (NB), which commonly occurs in the childhood. Development and application of a robust LC–MS/MS method for fast determination of these biomarkers for optimal laboratory testing of NB is essential in clinical laboratories. In present study, we developed and validated a simple liquid chromatography tandem mass spectrometry (LC–MS/MS) method for quick clinical testing of VMA and HVA for diagnosis of NB. The method was validated according to the current CLSI C62‐A and FDA guidelines. The age‐adjusted pediatric reference intervals and diagnostic performance were evaluated in both 24 h urine and random urine. Injection‐to‐injection time was 3.5 min. Inter‐ and intra‐assay coefficients of variation (CVs) were ≤3.88%. The lower limit of quantification and the limit of detection were 0.50 and 0.25 μmol/L for both VMA and HVA. Recoveries of VMA and HVA were in the ranges of 85–109% and 86–100% with CVs ≤5.76%. This method was free from significant matrix effect, carryover and interference. The establishment of age‐adjusted pediatric reference intervals by this LC–MS/MS method was favorable for the improvement in diagnostic performance, which was crucial for correct interpretation of test results from children in both 24 h and random urine.     

A sensitive LC–MS/MS method for the determination of triptolide and its application to pharmacokinetic research in rats

Triptolide is one of the main active ingredients of Tripterygium wilfordii Hook. F. In this study, a sensitive LC–MS/MS method was established and validated to determine the concentration of triptolide in rat plasma. Triptolide and an internal standard [(5R)‐5‐hydroxytriptolide] were extracted from 100 μL of rat plasma with acetonitrile, and the dried residue was then reconstituted and reacted with benzylamine to produce benzylamine triptolide and benzylamine (5R)‐5‐hydroxytriptolide. Derivatization increased the sensitivity of triptolide detection by ~100‐fold. Quantification was performed using a QTRAP 5500 tandem mass spectrometer with positive electrospray ionization in multiple reaction monitoring mode with an ion transition m/z 468.5 → 192.0 for benzylamine triptolide and m/z 484.3 → 192.1 for benzylamine (5R)‐5‐hydroxytriptolide. Good linearity was observed in the range of 0.030–100 ng/mL with a lower limit of quantitation of 0.030 ng/mL. The intra‐ and inter‐day precision was <6.5%, and the accuracy ranged from ?11.7 to ?4.4%. The recovery remained consistent and was reproducible at different concentrations. This method was successfully applied to the study of triptolide drug–drug interactions in Sprague–Dawley rats. With the use of itraconazole (40 mg/kg, p.o.) as a CYP3A inhibitor, the plasma exposure of triptolide in rats was increased by 36%.     

Development and validation of an LC–MS/MS method for the determination of hinokiflavone in rat plasma and its application to a pharmacokinetic study

Hinokiflavone has drawn a lot of attention for its multiple biological activities. In this study, a sensitive and selective method for determination of hinokiflavone in rat plasma was developed for the first time, using liquid chromatography–tandem mass spectrometry (LC–MS/MS). Amentoflavone was used as an internal standard. Separation was achieved on a Hypersil Gold C₁₈ column with isocratic elution using methanol–water (65:35, v /v) as mobile phase at a flow rate of 0.3 mL/min. A triple quadrupole mass spectrometer operating in the negative electrospray mode with selected reaction monitoring was used to detect the transitions of m/z 537 → 284 for hinokiflavone and m/z 537 → 375 for IS. The LOQ was 0.9 ng/mL with a linear range of 0.9–1000 ng/mL. The intra‐ and inter‐day accuracy (RE%) ranged from −3.75 to 6.91% and from −9.20 to 2.51% and the intra‐ and inter‐day precision (RSD) was between 0.32–14.11 and 2.85–10.04%. The validated assay was successfully applied to a pharmacokinetic study of hinokiflavone in rats. The half‐life of drug elimination at the terminal phase was 6.10 ± 1.86 h, and the area under the plasma concentration‐time curve from time zero to the time of last measurable concentration and to infinity values obtained were 2394.42 ± 466.86 and 2541.93 ± 529.85 h ng/mL, respectively.     

Use of high‐pH (basic/alkaline) mobile phases for LC–MS or LC–MS/MS bioanalysis

High‐pH or basic/alkaline mobile phases are not commonly used in LC–MS or LC–MS/MS bioanalysis because of the deeply rooted concern with column instability and reduced detection sensitivity for basic compounds in high‐pH mobile phases owing to charge neutralization. With the advancement of LC column technology and the wide recognition of the “wrong‐way‐round” phenomena, high‐pH mobile phases are more and more used in LC–MS or LC–MS/MS bioanalysis to improve chromatographic peak shape, retention, selectivity, resolution, and detection sensitivity, not only for basic compounds, but also for many other compounds. In this article, the benefits, practical considerations, application examples and cautions for using high‐pH mobile phases in LC–MS or LC–MS/MS bioanalysis are reviewed, with a focus on quantification. Furthermore, the future trends in this field are also envisaged. A total of 84 references are cited in this review.     

An LC–MS/MS method for rapid and sensitive high‐throughput simultaneous determination of various protein kinase inhibitors in human plasma

A reliable, high‐throughput and sensitive LC–MS/MS procedure was developed and validated for the determination of five tyrosine kinase inhibitors in human plasma. Following their extraction from human plasma, samples were eluted on a RP Luna®‐PFP 100 Å column using a mobile phase system composed of acetonitrile and 0.01 m ammonium formate in water (pH ~4.1) with a ratio of (50:50, v /v) flowing at 0.3 mL min⁻¹. The mass spectrometer was operating with electrospray ionization in the positive ion multiple reaction monitoring mode. The proposed methodology resulted in linear calibration plots with correlation coefficients values of r ² = 0.9995–0.9999 from concentration ranges of 2.5–100 ng mL⁻¹ for imatinib, 5.0–100 ng mL⁻¹ for sorafenib, tofacitinib and afatinib, and 1.0–100 ng mL⁻¹ for cabozantinib. The procedure was validated in terms of its specificity, limit of detection (0.32–1.71 ng mL⁻¹), lower limit of quantification (0.97–5.07 ng mL⁻¹), intra‐ and inter assay accuracy (−3.83 to +2.40%) and precision (<3.37%), matrix effect and recovery and stability. Our results demonstrated that the proposed method is highly reliable for routine quantification of the investigated tyrosine kinase inhibitors in human plasma and can be efficiently applied in the rapid and sensitive analysis of their clinical samples.     

Development and validation of a rapid LC‐MS/MS method to quantify letrozole in human plasma and its application to therapeutic drug monitoring

A selective, rapid, and sensitive liquid chromatography–tandem mass spectrometry(LC‐MS/MS) method was developed and validated for the determination of letrozole (LTZ) in human plasma, using anastrozole as internal standard (IS). Sample preparation was performed by one‐step protein precipitation with methanol. The analyte and IS were chromatographed on a reversed‐phase YMC‐ODS‐C₁₈ column (2.0 × 100 mm i.d., 3 µm) with a flow rate of 0.3 mL/min. The mobile phase consisted of water containing 0.1% formic acid (v/v) and methanol containing 0.1% formic acid (v/v). The mass spectrometer was operated in selected reaction monitoring mode through electrospray ionization ion mode using the transitions of m/z 286.2 → 217.1 for LTZ and m/z 294.1 → 225.1 for IS, respectively. The method was validated for selectivity, linearity, lower limit of quantitation, precision, accuracy, matrix effects and stability in accordance with the US Food and Drug Administration guidelines. Linear calibration curves were 1.0–60.0 ng/mL. Intra‐ and inter‐batch precision (CV) for LTZ were <9.34%, and the accuracy ranged from 97.43 to 105.17%. This method was successfully used for the analysis of samples from patients treated with LTZ in the dose of 2.5 mg/day. It might be suitable for therapeutic drug monitoring of these patients and contribute to predict the risk of adverse reactions. Copyright © 2015 John Wiley & Sons, Ltd.     

Simultaneous quantitation of metformin and dapagliflozin in human plasma by LC–MS/MS: Application to a pharmacokinetic study

A single LC–MS/MS assay has been developed and validated for the simultaneous determination of metformin and dapagliflozin in human plasma using ion‐pair solid‐phase extraction. Chromatographic separation of the analytes and their internal standards was carried out on a reversed‐phase ACE 5CN (150 × 4.6 mm, 5 μm) column using acetonitrile–15 mm ammonium acetate, pH 4.5 (70:30, v/v) as the mobile phase. To achieve higher sensitivity and selectivity for the analytes, mass spectrometric analysis was performed using a polarity switching approach. Ion transitions studied using multiple reaction monitoring mode were m/z 130.1 [M + H]⁺/60.1 for metformin and m/z 467.1 [M + CH₃COO]^?/329.1 for dapagliflozin in the positive and negative modes, respectively. The linear calibration range of the assay was established from 1.00 to 2000 ng/mL for metformin and from 0.10 to 200 ng/mL for dapagliflozin to achieve a better assessment of the pharmacokinetics of the drugs. The limit of detection and limit of quantitation for the analytes were 0.39 and 1.0 ng/mL for metformin and 0.03 and 0.1 ng/mL for dapagliflozin, respectively. There was no interference of plasma matrix obtained from different sources, including hemolyzed and lipemic plasma. The method was successfully applied to study the effect of food on the pharmacokinetics of metformin and dapagliflozin in healthy subjects.     

Analytical challenges in quantifying abiraterone with LC–MS/MS in human plasma

A method was developed and validated to quantify abiraterone in human plasma. During assay development, several analytical challenges were encountered: limited stability in patient samples, adsorption to glass, coelution with metabolites and carry‐over issues. Limited stability (2 h) was found for abiraterone in fresh plasma as well as whole blood at ambient temperature. When kept at 2–8°C, abiraterone in plasma was stable for 24 h and in whole blood for 8 h. Adsorption of abiraterone to glass materials was addressed by using polypropylene throughout the method. Carry‐over was reduced to acceptable limits by incorporating a third mobile phase into the gradient. The chromatographic separation of abiraterone with its multiple metabolites was addressed by using a longer analytical column and adjusting the gradient. Abiraterone was extracted by protein precipitation, separated on a C18 column with gradient elution and analyzed with tandem quadrupole mass spectrometry in positive ion mode. A stable deuterated isotope was used as the internal standard. The assay ranges from 1 to 500 ng/mL. Within‐ and‐between‐day precisions and accuracies were below 13.4% and within 95–102%. This bioanalytical method was successfully validated and applied to determine plasma concentrations of abiraterone in clinical studies and in regular patient care for patients with metastatic castration‐resistant prostate cancer.     

Determination of MLN0128, an investigational antineoplastic agent,in human plasma by LC–MS/MS

MLN0128, an mTOR kinase inhibitor, is currently undergoing clinical investigation for treatment of a variety of cancers. To support this work, an LC–MS/MS method has been developed for the determination of MLN0128 in human plasma. A structural analog STK040263 was used as the internal standard. Both MLN0128 and the IS were first extracted from plasma using methyl tert ‐butyl ether; then separated on a Waters XTerra® MS C₁₈ column using a mobile phase consisting of methanol–acetonitrile–10.0 mm ammonium formate (34:6:60, v /v/v) at a flow rate of 0.300 mL min⁻¹. Quantitation of MLN0128 was done by positive electrospray ionization tandem mass spectrometry in multiple‐reaction‐monitoring mode. This method has a total run time of <4 min with the retention times of 1.95 and 2.94 min for the IS and MLN0128, respectively. The method has been validated per the US Food and Drug Administration guidance for bioanalytical method validation. It has a calibration range of 0.100–50.0 ng mL⁻¹ in human plasma with a correlation coefficient > 0.999. The overall assay accuracy and precision were ≤ ± 4 and ≤8%, respectively. The IS normalized recovery of MLN0128 was 98–100%. The stability studies showed that MLN0128 was stable under all tested conditions. The method developed may be useful for clinical studies of MLN0128.     

Development of an LC–MS/MS method for quantification of two isomeric phenylpropenes and the application to pharmacokinetic studies in rats

Isomers β‐asarone and α‐asarone have recently been demonstrated to have differential pharmacological activities . Here, we report an LC–MS/MS method developed using acetonitrile to extract two isomeric phenylpropenes from rat plasma. Separation was achieved using a XDB‐C₁₈ column (100 × 2.1 mm; i.d., 1.8 μm) with a mobile phase of methanol–0.1% formic acid (55:45, v/v) at a flow rate of 0.3 mL/min. Calibration curves ranging from 5.20 to 2080 ng/mL for β‐asarone and from 3.68 to 1470 ng/mL for α‐asarone were linear (r² ≥ 0.9938) with the lower limits of quantification being 5.20 and 3.68 ng/mL for both isomers. Intravenous administration of β‐asarone (2.22 mg/kg) and α‐asarone (2.36 mg/kg) in rats yielded half‐lives of 13.40 ± 4.11 and 28.88 ± 7.82 min with clearance values of 0.196 ± 0.062 mL/min/kg and 0.112 ± 0.012 mL/min/kg for β‐asarone and α‐asarone, respectively.     

Development and application of a LC–MS/MS assay for the simultaneous quantification of edaravone and taurine in beagle plasma

An LC–MS/MS method was developed and validated for the simultaneous quantification of edaravone and taurine in beagle plasma. The plasma sample was deproteinized using acetonitrile containing formic acid. Chromatographic separations were achieved on an Agilent Zorbax SB‐Aq (100 × 2.1 mm, 3.5 μm) column, with a gradient of water (containing 0.03% formic acid) and methanol as the mobile phase at a flow rate of 0.3 mL/min. The analyte detection was carried out in multiple reaction monitoring mode and the optimized precursor‐to‐product transitions of m/z [M+H]⁺ 175.1 → 133.0 (edaravone), m/z [M+H]⁺ 189.1 → 147.0 (3‐methyl‐1‐p‐tolyl‐5‐pyrazolone, internal standard, IS), m/z [M–H]^? 124.1→80.0 (taurine), and m/z [M–H]^? 172.0 → 80.0 (sulfanilic acid, IS) were employed to quantify edaravone, taurine, and their corresponding ISs, respectively. The LOD and the lower LOQ were 0.01 and 0.05 μg/mL for edaravone and 0.66 and 2 μg/mL for taurine, respectively. The calibration curves of these two analytes demonstrated good linearity (r ＞ 0.99). All the validation data including the specificity, precision, recovery, and stability conformed to the acceptable requirements. This validated method has successfully been applied in the pharmacokinetic study of edaravone and taurine mixture in beagle dogs.     

Comparative pharmacokinetic study on three formulations of Astragali Radix by an LC–MS/MS method for determination of formononetin in human plasma

Astragali Radix (AR) is a widely used traditional Chinese medicine for healing the cardiovascular, liver and immune systems. Recently, superfine pulverizing technology has been applied to developing novel formulations to improve bioavailability of the active constituents in herbs, such as ultrafine granular powder of AR. In this study, a universal and sensitive quantitative method based on LC–MS/MS was employed for determining formononetin, the main flavonoid in AR, in human plasma for comparative pharmacokinetics of three oral formulations of AR. Formononetin and IS (quercetin) were extracted by ethyl acetate from human plasma and were separated on a C₁₈ column with a mobile phase consisting of acetonitrile and 0.1% formic acid. Positive‐ion electrospray‐ionization mode was applied in mass spectrometric detection. The quantitative method was validated with regards to selectivity, linearity, accuracy and precision, matrix effect, extraction recovery and stability, and was applied to comparing the pharmacokinetics of ultrafine granular powder (UGP), ultrafine powder (UP) and traditional decoction pieces (TDP) of AR after oral administration. The peak concentration and areas under the concentration–time curve of formononetin in UGP and UP were significantly higher than those of TDP. UGP and UP could significantly improve the bioavailability of AR in human compared with TDP after oral administration.     

Simultaneous LC–MS/MS analysis of eicosanoids and related metabolites in human serum,sputum and BALF

The differences among individual eicosanoids in eliciting different physiological and pathological responses are largely unknown because of the lack of valid and simple analytical methods for the quantification of individual eicosanoids and their metabolites in serum, sputum and bronchial alveolar lavage fluid (BALF). Therefore, a simple and sensitive LC–MS/MS method for the simultaneous quantification of 34 eicosanoids in human serum, sputum and BALF was developed and validated. This method is valid and sensitive with a limit of quantification ranging from 0.2 to 3 ng/mL for the various analytes, and has a large dynamic range (500 ng/mL) and a short run time (25 min). The intra‐ and inter‐day accuracy and precision values met the acceptance criteria according to US Food and Drug Administration guidelines. Using this method, detailed eicosanoid profiles were quantified in serum, sputum and BALF from a pilot human study. In summary, a reliable and simple LC–MS/MS method to quantify major eicosanoids and their metabolites was developed and applied to quantify eicosanoids in human various fluids, demonstrating its suitability to assess eicosanoid biomarkers in human clinical trials.     

Simultaneous quantitation of rosuvastatin and ezetimibe in human plasma by LC–MS/MS: Pharmacokinetic study of fixed‐dose formulation and separate tablets

A simple, high‐throughput and highly sensitive liquid chromatography coupled with tandem mass spectrometry (LC–MS/MS) method has been developed for the simultaneous estimation of rosuvastatin and free ezetimibe. Liquid–liquid extraction was carried out using methyl‐tert butyl ether after prior acidification from 300 μL human plasma. The recovery for both the analytes and their deuterated internal standards (ISs) ranged from 95.7 to 99.8%. Rosuvastatin and ezetimibe were separated on Symmetry C₁₈ column using acetonitrile and ammonium formate buffer, pH 3.5 (30:70, v/v) as the mobile phase. The analytes were well resolved with a resolution factor of 3.8. Detection and quantitation were performed under multiple reaction monitoring using ESI(+) for rosuvastatin (m/z 482.0 → 258.1) and ESI(−) for ezetimibe (m/z 407.9 → 271.1). A linear response function was established in the concentration ranges of 0.05–50.0 ng/mL and 0.01–10.0 ng/mL for rosuvastatin and ezetimibe, respectively, with correlation coefficient, r² ≥ 0.9991. The IS‐normalized matrix factors for the analytes ranged from 0.963 to 1.023. The developed method was successfully used to compare the pharmacokinetics of a fixed‐dose combination tablet of rosuvastatin‐ezetimibe and co‐administered rosuvastatin and ezetimibe as separate tablets to 24 healthy subjects. The reliability of the assay was also assessed by reanalysis of 115 subject samples.     

Application of a LC–MS/MS method developed for the determination of p‐phenylenediamine,N‐acetyl‐p‐phenylenediamine and N,N‐diacetyl‐p‐phenylenediamine in human urine specimens

Cases of poisoning by p‐phenylenediamine (PPD) are detected sporadically. Recently an article on the development and validation of an LC–MS/MS method for the detection of PPD and its metabolites, N‐acetyl‐p‐phenylenediamine (MAPPD) and N,N‐diacetyl‐p‐phenylenediamine (DAPPD) in blood was published. In the current study this method for detection of these compounds was validated and applied to urine samples. The analytes were extracted from urine samples with methylene chloride and ammonium hydroxide as alkaline medium. Detection was performed by LC–MS/MS using electrospray positive ionization under multiple reaction‐monitoring mode. Calibration curves were linear in the range 5–2000 ng/mL for all analytes. Intra‐ and inter‐assay imprecisions were within 1.58–9.52 and 5.43–9.45%, respectively, for PPD, MAPPD and DAPPD. Inter‐assay accuracies were within ?7.43 and 7.36 for all compounds. The lower limit of quantification was 5 ng/mL for all analytes. The method, which complies with the validation criteria, was successfully applied to the analysis of PPD, MAPPD and DAPPD in human urine samples collected from clinical and postmortem cases.     

Determination of ospemifene in human plasma by LC–MS/MS and its application to a human pharmacokinetic study

A highly sensitive, specific and rapid liquid chromatography–tandem mass spectrometry (LC–MS/MS) analytical method has been developed and validated for the determination of ospemifene in human plasma using ospemifene‐d₄ as an internal standard. Solid‐phase extraction technique with Phenomenex Strata X‐33 μm polymeric sorbent cartridges (30 mg/1 mL) was used to extract the analytes from the plasma. The chromatographic separation was achieved on Agilent Eclipse XDB‐Phenyl, 4.6 × 75 mm, 3.5 μm column using the mobile phase composition of methanol and 20 mm ammonium formate buffer (90:10, v/v) at a flow rate of 0.9 mL/min. A detailed method validation was performed as per the US Food and Drug Administration guidelines and the calibration curve obtained was linear (r² = 99) over the concentration range 5.02–3025 ng/mL. The API‐4500 MS/MS was operated under multiple reaction monitoring mode during the analysis. The proposed method was successfully applied to a pharmacokinetic study in healthy human volunteers after oral administration of an ospemifene 60 mg tablet under fed conditions.