Determination of ticagrelor and two metabolites in plasma samples by liquid chromatography and mass spectrometry

Rapid and sensitive analytical methods using liquid chromatography with tandem mass spectrometry (LC/MS/MS) were developed for the determination of ticagrelor, the first reversible oral platelet P2Y₁₂ receptor inhibitor, and its metabolites AR-C124910XX and AR-C133913XX in human plasma. Ticagrelor and its metabolites were extracted using protein precipitation with acetonitrile. Chromatographic separations were performed on reversed phase columns and detection using atmospheric pressure chemical ionization (APCI). Ticagrelor and AR-C124910XX were analyzed in the same assay, with the internal standard, d7-ZD6140, on a C18 column using negative ionization; AR-C133913XX analyzed separately on a phenyl column using positive ionization. Full validation of the methods was performed including selectivity, lower limit of quantification, accuracy, precision stability and incurred sample reproducibility and incurred sample stability. Total analytical run time was short (2 min). Calibration curves were established in the range 5–5000 ng/mL for ticagrelor, 2.5–2500 ng/mL for AR-C124910XX and 2–1000 ng/mL for AR-C133913XX. Lower limits of quantification for ticagrelor, AR-C124910XX and AR-C133913XX were determined to be 5, 2.5 and 2.0 ng/mL, respectively from 100 μL of human plasma. For ticagrelor, AR-C124910XX and AR-C133913XX, mean intra-batch accuracy was 91.9–109.0%, 86.8–109.2% and 100.5–112.0%, respectively; intra-batch precision was 4.0–8.4%, 5.2–16.9% and 3.9–12.3%, respectively. The methods were also applied to quantification of ticagrelor, AR-C124910XX and AR-C133913XX in rabbit, rat, mouse and marmoset, using 25 μL of animal plasma. A modified methodology was developed to quantify ticagrelor and AR-C124910XX in plasma from dog and cynomolgus monkey. Human incurred samples were found to generate consistent reproducibility and stability results. This method was successfully applied to determine plasma concentrations following administration of ticagrelor in human volunteers and patients, and animal safety evaluation studies. This validated methods has the advantages of being straightforward, robust and allows a fast throughput of samples.     

Determination of sitagliptinin human plasma using protein precipitation and tandem mass spectrometry

A simple offline LC–MS/MS method for the quantification of sitagliptin in human plasma is described. Samples are prepared using protein precipitation. Filtration of the supernatants through a Hybrid-SPE-PPT plate was found to be necessary to reduce ionization suppression caused by co-elution of phospholipids with sitagliptin. The sitagliptin and its stable isotope labeled internal standard (IS) were chromatographed under hydrophilic interaction chromatography conditions on a Waters Atlantis HILIC Silica column (2.1 mm × 50 mm, 3 μm) using a mobile phase of ACN/H₂O (80/20, v/v) containing 10 mM NH₄Ac (pH 4.7). The sample drying after protein precipitation due to high organic content in the sample is not necessary, because HILIC column was used. The analytes were detected with a tandem mass spectrometer employing a turbo ion spray (TIS) interface in positive ionization mode. The multiple reaction monitoring (MRM) transitions were m/z 408 → 235 for sitagliptin and m/z 412 → 239 for IS. The lower limit of quantitation (LLOQ) for this method is 1 ng/mL when 100 μL of plasma is processed. The linear calibration range is 1–1000 ng/mL for sitagliptin. Intra-day precision and accuracy were assessed based on the analysis of six sets of calibration standards prepared in six lots of human control plasma. Intra-day precision (RSD%, n = 6) ranged from 1.2% to 6.1% and the intra-day accuracy ranged from 97.6% to 103% of nominal values.     

Simple,sensitive and rapid HPLC–MS/MS method for the determination of cepharanthine in human plasma

A rapid, sensitive and specific method for the determination of cepharanthine in human plasma using high performance liquid chromatography coupled with tandem mass spectrometry (HPLC–MS/MS) was described. Cepharanthine and the internal standard (I.S.), telmisartan, were extracted from human plasma by methanol to precipitate the protein. A centrifuged upper layer was then evaporated and reconstituted with 100 μL methanol. Chromatographic separation was performed on an AGILENT XDB-C₈ column (150 mm × 2.1 mm, 5.0 μm, Agilent, USA) using a gradient mobile phase with 1 mmol/L ammonium acetate in water with 0.05% formic acid and methanol. Detection and quantitation was performed by MS/MS using electrospray ionization (ESI) and multiple reaction monitoring (MRM) in the positive ion mode. The most intense [M+H]⁺ MRM transition of cepharanthine at m/z 607.3 → 365.3 was used for quantitation and the transition at m/z 515.5 → 276.4 was used to monitor telmisartan. The calibration curve was linear within the concentration range of 0.5–200.0 ng/mL (r = 0.9994). The limit of quantification (LOQ) was 0.5 ng/mL. The extraction recovery was above 81.1%. The accuracy was higher than 92.3%. The intra- and inter-day precisions were less than 9.66%. The method was accurate, sensitive and simple and was successfully applied to a pharmacokinetic study after single intravenous administration of 50 mg cepharanthine in 12 healthy Chinese volunteers.     

Quantitative analysis of lignocaine and metabolites in equine urine and plasma by liquid chromatography–tandem mass spectrometry

In this paper, a method for the sensitive and reproducible analysis of lignocaine and its four principal metabolites, monoethylxylidide (MEGX), glycylxylidide (GX), 3-hydroxylignocaine (3-HO-LIG), 4-hydroxylignocaine (4-HO-LIG) in equine urine and plasma samples is presented. The method uses liquid chromatography coupled to tandem mass spectrometry operating in electrospray ionisation positive ion mode (+ESI) via multiple reaction monitoring (MRM). Sample preparation involved solid-phase extraction using a mixed-mode phase. The internal standard adopted was lignocaine-d₁₀. Lignocaine and its metabolites were successfully resolved using an octadecylsilica reversed-phase column using a gradient mobile phase of acetonitrile and 0.1% (v/v) aqueous formic acid at a flow rate of 300 μL/min. Target analytes and the internal standard were determined by using the following transitions; lignocaine, 235.2 > 86.1; 3-HO-LIG and 4-HO-LIG, 251.2 > 86.1; MEGX, 207.1 > 58.1; GX, 179.1 > 122.1; and lignocaine-d₁₀, 245.2 > 96.1. Calibration curves were generated over the range 1–100 ng/mL for plasma samples and 1–1000 ng/mL for urine samples. The method was validated for instrument linearity, repeatability and detection limit (IDL), method linearity, repeatability, detection limit (MDL), quantitation limit (LOQ) and recovery. The method was successfully used to analyse both plasma and urine samples following a subcutaneous administration of lignocaine to a thoroughbred horse.     

Development and validation of a liquid chromatography–mass spectrometry method for the determination of verticinone in rat plasma and its application to pharmacokinetic study

We have developed and validated a sensitive liquid chromatography–electrospray ionization-mass spectrometric (LC–ESI-MS) method for the quantification of verticinone, a major active constituent from Fritillaria hupehensis Hsiao et KC Hsia., in rat plasma. Verticinone and the internal standard (IS), hupehenine, were extracted from plasma samples by a simple liquid–liquid extraction with ethyl acetate after being alkalified by 1 M ammonia hydroxide. Chromatographic separation was achieved on a C₁₈ column using a gradient elution program with methanol and water as the mobile phase. The detection was performed by selected ion monitoring (SIM) mode via positive electrospray ionization (ESI) interface. The lower limit of quantification (LLOQ) was 0.1 ng/mL. The calibration curves were linear (r² > 0.998) over the concentration range of 0.1–200 ng/mL. Within- and between-run precision was less than 6.5% and accuracy was within ±10.7%. The validated method was applied to the pharmacokinetic study of verticinone in rats after a single oral administration of 1 mg/kg.     

Quantitative analysis of 3α,6α,24-trihydroxy-24,24-di(trifluoromethyl)-5β-cholane,a potent synthetic steroidal liver X receptor agonist in plasma using liquid chromatography–tandem mass spectrometry

The steroidal liver X receptor agonist, 3α,6α,24-trihydroxy-24,24-di(trifluoromethyl)-5β-cholane (ATI-829) is a potential therapeutic agent for the treatment of atherosclerosis. A sensitive and selective liquid chromatography–tandem mass spectrometry (LC–MS–MS) method for the quantification of ATI-829 in mouse plasma was developed and validated. Proteins in a 25 μL aliquot of mouse plasma were precipitated, and ATI-829 was extracted from the precipitate by the addition of 125 μL methanol. The overall extraction efficiency was greater than 99%. LC–MS–MS with negative ion electrospray and selected reaction monitoring was used for the quantitative analysis of ATI-829. The lower limit of quantitation of ATI-829 corresponded to 5.0 ng/mL (9.7 nM) plasma. Interference from matrix was negligible. The calibration curve was linear over the range 5–2000 ng/mL. The intra-day precision and inter-day precision of the analyses were <4.5% and <6%, respectively, and the accuracy ranged from 92% to 103%. ATI-829 in plasma was stable for at least 6 h at room temperature, 1 week at 4 °C, and 3 weeks at −20 °C. The validated method was then utilized for pharmacokinetic studies of ATI-829 administered to mice.     

Development and validation of an HPLC–FLD method for milbemectin quantification in dog plasma

Milbemectin is a widely used veterinary antiparasitic agent. A high-performance liquid chromatography with fluorescent detection (HPLC–FLD) method is described for the determination of milbemectin in dog plasma. The derivative procedure included mixing 1-methylimizole [MI, MI-ACN (1:1, v/v), 100 μL], trifluoroacetic anhydride [TFAA, TFAA-ACN (1:2, v/v), 150 μL] with a subsequent incubation for 3 s at the room temperature to obtain a fluorescent derivative, which is reproducible in different blood samples and the derivatives proved to be stable for at least 80 h at room temperature. HPLC method was developed on C18 column with FLD detection at an excitation wavelength of 365 nm and emission wavelength of 475 nm, with the mobile phase consisting of methanol and water in the ratio of 98:2 (v/v). The assay lower limit of quantification was 1 ng/mL. The calibration curve was linear over concentration range of 1–200 ng/mL. The intra- and inter-day accuracy was >94% and precision expressed as % coefficient of variation was <5%. This method is specific, simple, accurate, precise and easily adaptable to measure milbemycin in blood of other animals.     

Enantioselective determination of doxazosin in human plasma by liquid chromatography–tandem mass spectrometry using ovomucoid chiral stationary phase

An enantioselective and sensitive method was developed and validated for determination of doxazosin enantiomers in human plasma by liquid chromatography–tandem mass spectrometry. The enantiomers of doxazosin were extracted from plasma using ethyl ether/dichloromethane (3/2, v/v) under alkaline conditions. Baseline chiral separation was obtained within 9 min on an ovomucoid column using an isocratic mobile phase of methanol/5 mM ammonium acetate/formic acid (20/80/0.016, v/v/v) at a flow rate of 0.60 mL/min. Acquisition of mass spectrometric data was performed in multiple reaction monitoring mode, using the transitions of m/z 452 → 344 for doxazosin enantiomers, and m/z 384 → 247 for prazosin (internal standard). The method was linear in the concentration range of 0.100–50.0 ng/mL for each enantiomer using 200 μL of plasma. The lower limit of quantification (LLOQ) for each enantiomer was 0.100 ng/mL. The intra- and inter-assay precision was 5.0–11.1% and 5.7–7.6% for R-(−)-doxazosin and S-(+)-doxazosin, respectively. The accuracy was 97.4–99.5% for R-(−)-doxazosin and 96.8–102.8% for S-(+)-doxazosin. No chiral inversion was observed during the plasma storage, preparation and analysis. The method proved adequate for enantioselective pharmacokinetic studies of doxazosin after oral administration of therapeutic doses of racemic doxazosin.     

Déficit de vitamina D en una cohorte de mayores de 65 años: prevalencia y asociación con factores sociodemográficos y de salud

Introduction

Vitamin D deficiency is common in the elderly, especially among institutionalized and/or hip fracture patients. However, there are few population studies on the prevalence of this deficiency in the general population over 64 years in our environment. The aim of this study was to determine the prevalence of vitamin D deficiency in an urban population cohort of over 64 years, and analyze its relationship with sociodemographic, climatic, and health factors.

Material and methods

Cross-sectional study from «Peñagrande cohort», a population-based cohort consisting of people over 64 years. We determined 25-hydroxyvitamin D levels, and recorded sociodemographic data (age, sex, marital status, education, socioeconomic status), season of measurement and health variables (comorbidity, obesity, malnutrition, renal failure, cognitive impairment, vitamin D supplements, and disability).

Results

A total of 468 individuals with a mean age of 76.0 years (SD: 7.7) were included, of which 53.4% were women. The mean value of vitamin D was 20.3 ± 11.7 ng/mL. The large majority (86.3%, 95% CI: 83.0-89.5) had a vitamin insufficiency (≤ 30 ng/ml), and 35.2% (95% CI: 30.8-39.7) showed severe vitamin deficiency (≤ 15 ng/ml). Vitamin insufficiency increases linearly with age (OR 1.06; 95% CI: 1.01-1.11), and was associated with low socioeconomic status (OR 3.29; 95% CI: 1.55-6.95). Severe vitamin D deficiency increases with age (OR 1.06; 95% CI: 1.02-1.09), female gender (OR 1.80; 95% CI: 1.18-2.75) and with cognitive impairment (OR 1.71; 95% CI: 1.04-2.83).

Conclusion

The prevalence of vitamin D deficiency in people over 65 years of age in our community is high. It would be advisable to determine the vitamin D values in the high risk elderly in order to introduce measures of pharmacological supplementation in those with inadequate levels.     

Development and validation of a liquid chromatography–tandem mass spectrometry method for the determination of goserelin in rabbit plasma

A rapid and sensitive liquid chromatography–electrospray ionization tandem mass spectrometry method (LC–ESI-MS/MS) was developed and validated for the determination of goserelin in rabbit plasma. Various parameters affecting plasma sample preparation, LC separation, and MS/MS detection were investigated, and optimized conditions were identified. Acidified plasma samples were applied to Oasis^® HLB solid-phase extraction (SPE) cartridges. Extracted samples were evaporated under a stream of nitrogen and then reconstituted with 100 μL mobile phase A. The separation was achieved on a Capcell-Pak C18 (2.0 mm × 150 mm, 5 μm, AQ type) column with a gradient elution of solvent A (0.05% acetic acid in deionized water/acetonitrile = 85/15; v/v) and solvent B (acetonitrile) at a flow rate of 250 μL/min. The LC–MS/MS system was equipped with an electrospray ion source operating in positive ion mode. Multiple-reaction monitoring (MRM) of the precursor–product ion transitions consisted of m/z 635.7 → m/z 607.5 for goserelin and m/z 424.0 → m/z 292.1 for cephapirin (internal standard). The proposed method was validated by assessing specificity, linearity, limit of quantification (LOQ), intra- and inter-day precision and accuracy, recovery, and stability. Linear calibration curves were obtained in the concentration range of 0.1–20 ng/mL (the correlation coefficients were above 0.99). The LOQ of the method was 0.1 ng/mL. Results obtained from the validation study of goserelin showed good accuracy and precision at concentrations of 0.1, 1, 5, 10, and 20 ng/mL. The validated method was successfully applied to a pharmacokinetic study of goserelin after a single subcutaneous injection of 3.6 mg of goserelin in healthy white rabbits.     

Development and validation of sensitive and selective LC–MS/MS methods for the determination of BMS-708163, a γ-secretase inhibitor,in plasma and cerebrospinal fluid using deprotonated or formate adduct ions as precursor ions

BMS-708163 is a γ-secretase inhibitor that is being developed for the treatment of Alzheimer's disease. Several LC–MS/MS methods have been developed for the determination of BMS-708163 in both plasma and cerebrospinal fluid in support of dog, rat, mouse and human studies. To support non-clinical studies, an LC–MS/MS method with a lower limit of quantitation (LLOQ) of 5 ng/mL, was developed and validated in dog, rat, and mouse plasma by using the deprotonated ion as the precursor ion. To support clinical studies, an LC–MS/MS method with LLOQ of 0.1 ng/mL, was developed and validated in human plasma by using the formate adduct as the precursor ion. Formic acid (0.01%) in water and acetonitrile was found to be the most favorable mobile phases for both deprotonated and formate adduct ions in negative electrospray ionization mode. A combination of a 3M Empore™ C18 plate for SPE and a Waters Atlantis dC18 analytical column for separation was used to achieve a highly selective solid phase extraction and chromatographic procedure from plasma without dry down and reconstitution steps. In the development of an assay for BMS-708163 in cerebral spinal fluid (CSF), significant non-specific binding of BMS-708163 was observed and resolved with pre- or post-spike of 0.2% Tween 20 into CSF samples. A dilute-and-shoot LC–MS/MS method with LLOQ of 0.1 ng/mL was developed and validated to assess BMS-708163 exposure in human CSF.     

A fast and sensitive HPLC–MS/MS analysis and preliminary pharmacokinetic characterization of ergone in rats

A fast and sensitive HPLC–APCI-MS/MS method was developed for the determination of ergosta-4,6,8(14),22-tetraen-3-one (ergone) in rat plasma. The plasma sample containing ergone and ergosterol (internal standard) were simply treated with acetone to precipitate and remove proteins and the isolated supernatants were directly injected into the HPLC–APCI-MS/MS system. Chromatographic separation was performed on a 1.8 μm Zorbax SB-C18 column (100 mm × 3.0 mm) with a 97:3 (v/v) mixed solution of methanol and 0.1% aqueous formic acid being used as mobile phase. Quantification was performed by multiple selected reactions monitoring (MRM) of the transitions with (m/z)⁺ 393–268 for ergone and (m/z)⁺ 379–69 for the IS. The method was validated in the concentration range of 5–1600 ng/mL for ergone. The precision of the assay (RSD%) was less than 10.5% at all concentrations levels within the tested range and adequate accuracy, and the limit of detection was 1.5 ng/mL. The absolute recoveries of both ergone and ergosterol from the plasma were more than 95%. The developed method has been successfully applied to the pharmacokinetic study of the drug in SD rats.     

Improved liquid chromatography–tandem mass spectrometry method for the analysis of eptifibatide in human plasma

A rapid, selective and highly sensitive high performance liquid chromatography–tandem mass spectrometry method (LC–MS/MS) was developed and validated for the determination and pharmacokinetic investigation of eptifibatide in human plasma. Eptifibatide and the internal standard (IS), EPM-05, were extracted from plasma samples using solid phase extraction. Chromatographic separation was performed on a C₁₈ column at a flow rate of 0.5 mL/min. Detection of eptifibatide and the IS was achieved by tandem mass spectrometry with an electrospray ionization (ESI) interface in positive ion mode. Traditional multiple reaction monitoring (MRM) using the transition of m/z 832.6 → m/z 646.4 and m/z 931.6 → m/z 159.4 was performed to quantify eptifibatide and the IS, respectively. The calibration curves were linear over the range of 1–1000 ng/mL with the lower limit of quantitation validated at 1 ng/mL. The intra- and inter-day precisions were within 13.3%, while the accuracy was within ±7.6% of nominal values. The validated LC–MS/MS method was successfully applied for the evaluation of pharmacokinetic parameters of eptifibatide after intravenous (i.v.) administration of a 45 μg/kg bolus of eptifibatide to 8 healthy volunteers.     

Determination of puerarin in rat plasma by rapid resolution liquid chromatography tandem mass spectrometry in positive ionization mode

A highly sensitive and specific method of rapid resolution liquid chromatography tandem mass spectrometry (RRLC-MS/MS) in positive ionization mode has been developed and validated for pharmacokinetic study of puerarin in rat plasma. Chromatography was carried out on a Zorbax XDB C18 reversed-phase column using a mobile phase comprising a mixture of methanol and 0.05% acetic acid in water (35:65, v/v) with a flow rate of 0.3 mL/min from 0 min to 5.4 min and then 0.6 mL/min from 5.41 min to 12 min. The mass spectrometer operated in ESI positive ionization mode. Multiple reaction monitoring (MRM) was used to measure puerarin and tectoridin (internal standard). The method was sensitive with a detection limit of 0.33 ng/mL. A good linear response was observed over a range of 10-2000 ng/mL in rat plasma. The inter- and intra-day precision ranged from 2.97% to 7.52% and accuracy from 93.70% to 101.60%. This validated method was applied successfully to a pharmacokinetic study in rat plasma after intravenous administration of puerarin. The main pharmacokinetic parameters were as follows: AUC(0→t) 45.37±13.19 (mgh/L), AUC(0→∞) 47.03±14.78 (mgh/L), MRT 1.03±0.46 (h), T(1/2) 1.31±0.31 (h), V(ss) 0.09±0.02 (L), V(z) 0.17±0.04 (L), Cl 0.10±0.04 (L/h).     

A fast and sensitive HPLC-MS/MS analysis and preliminary pharmacokinetic characterization of cudratricusxanthone B in rats

A method for the quantitative analysis of cudratricusxanthone B (CXB) in rat plasma by high performance liquid chromatography–electrospray ionization-tandem mass spectrometry (HPLC–ESI-MS/MS) has been developed and validated. The method involved liquid–liquid extraction from plasma, simple chromatographic conditions on a Venusil XBP-PH C₁₈ column with the mobile phase of 0.5% formic acid in methanol, and mass spectrometric detection using an API-3000 instrument. Multiple reaction monitoring (MRM) mode was used to monitor precursor/product ion transitions of m/z 397.1/285.0 for CXB and m/z 381.6/269.2 for the internal standard (I.S.) cudraxanthone H. The standard curves were linear over the concentration range of 1–500 ng/mL for CXB in rat plasma. The intra- and inter-batch accuracy for CXB at four concentrations was 89.4–99.5% and 89.4–100.8%, respectively. The RSDs were less than 7.92%. The lower limit of quantification for CXB was 1.0 ng/mL using 100 μL of plasma. The average extraction recoveries of CXB ranged from 80.1 to 95.4% at the concentrations of 2, 50 and 500 ng/mL, respectively. This method was successfully applied to the pharmacokinetic study after an intravenous administration of CXB in male Sprague–Dawley (SD) rats.     

Measurement of cortisol,cortisone, prednisolone,dexamethasone and 11-deoxycortisol with ultra high performance liquid chromatography–tandem mass spectrometry: Application for plasma,plasma ultrafiltrate,urine and saliva in a routine laboratory

We describe an ultra high performance liquid chromatography–tandem mass spectrometry (UHPLC MS/MS) method suitable for a routine laboratory to determine endogenous and exogenous glucocorticoids in plasma, plasma ultrafiltrate, urine and saliva in a single analytical run. After addition of a multi-analyte internal standard, a standardised sample preparation procedure with solid phase extraction followed, before injecting into a tandem mass spectrometer with positive mode electron spray ionisation and multiple reactant monitoring acquisition. The chromatography time was 3 min. The limit of quantitation for cortisol and cortisone in plasma was 3.75 nmol/L and linearity extended to 2000 nmol/L. The limit of quantitation for cortisol in plasma ultrafiltrate and saliva was 0.6 nmol/L. The limit of quantitation for 11-deoxycortisol and prednisolone was 5 nmol/L and for dexamethasone 1 nmol/L. The intra-assay CV was <5% and the inter-assay CV <10% for all analytes in all matrices. Comparison with an immuno-assay (IA) plasma cortisol method resulted in a regression equation of UHPLC = 0.79 × IA + 31.12 with R² = 0.960 (p < 0.0001). Comparison with a high performance liquid chromatography (HPLC) cortisol method yielded a regression equation of UHPLC = 1.06 × HPLC + 9.82, R² = 0.992 (p < 0.0001). The simultaneous measurement of endogenous and exogenous glucocorticoids contributed to patient care in cases with dexamethasone and metyrapone dynamic tests and unsuspected therapeutic glucocorticoid use.     

Validation of a total testosterone assay using high-turbulence liquid chromatography tandem mass spectrometry: Total and free testosterone reference ranges

Accurate measurement of testosterone concentration is of critical importance when diagnosing and treating male hypogonadism, congenital adrenal hyperplasia, premature or delayed puberty, and androgen excess in polycystic ovary syndrome or other virilizing conditions. However, some assays have inherent limitations and biases that affect measurement of low-testosterone values. Therefore, we developed a highly specific online mass spectrometry method. Sera were extracted online using high-turbulence flow liquid chromatography coupled to analytical HPLC and atmospheric pressure chemical ionization tandem mass spectrometry (HTLC-APCI-MS/MS). Analyte ions were monitored by multiple reaction monitoring (MRM). Total analysis time was 1.15 min per sample when using the multiplexing system. Testosterone concentrations were measured directly from 150 μL of serum or plasma without derivatization or liquid-liquid extraction. The lower limit of quantification was 0.3 ng/dL, and the assay was linear up to 2000 ng/dL. The method compared very well with an established RIA: y = 1.02x + 1.5, r² = 0.994. Comparison with a platform immunoassay confirmed the previously reported ICMA positive bias at low concentrations. Male and female adult and pediatric reference ranges were developed for this very sensitive and accurate high-throughput LC-MS/MS method. This method is suitable for measuring the expected low-testosterone concentrations seen in women, children, and hypogonadal males and for monitoring testosterone suppressive therapy in prostate cancer patients.     

Sensitive and rapid quantification of the cannabinoid receptor agonist naphthalen-1-yl-(1-pentylindol-3-yl)methanone (JWH-018) in human serum by liquid chromatography–tandem mass spectrometry

The current paper describes a validated method for the detection and quantification of naphthalen-1-yl-(1-pentylindol-3-yl)methanone (JWH-018), an ingredient of a herbal mixture called “Spice”, by means of HPLC–ESI–MS–MS in serum. Lower limit of detection and lower limit of quantification were 0.07 and 0.21 ng/ml, respectively. In 2 subjects who consumed ca. 50 μg/kg of JWH-018 by smoking, the active ingredient was detected by means of the described method. Thereby, the serum concentrations reached values of approx. 10 ng/ml and dropped within 3 h very fast (<10% of the measured maximum concentrations).     

Ferrocene-based Diels-Alder derivatization for the determination of 1alpha-hydroxyvitamin D3 in rat plasma by high-performance liquid chromatography-electrospray tandem mass spectrometry

A specific and sensitive liquid chromatography-electrospray ionization tandem mass spectrometry (LC/ESI-MS/MS) method has been developed for the determination of 1alpha-hydroxyvitamin D(3) (1alphaOHD(3)) in rat plasma. A new ferrocene-based Cookson-type reagent, 4-ferrocenylmethyl-1,2,4-triazoline-3,5-dione (FMTAD), designed and synthesized to be highly sensitive to vitamin D analogs in ESI, considerably improved the detection limit with 250 fg (359 amol)/injection. 1alphaOHD(3) in rat plasma was extracted with acetonitrile and then purified using Oasis HLB 96-well plates. After the precolumn derivatization with FMTAD, samples were subjected to LC/ESI-MS/MS employing a column-switching system. This method achieved a lower limit of quantitation of 5 pg from 0.1-mL plasma aliquots and 200-fold sensitivity of that without derivatization. The calibration curve (0.05-15 ng/mL) exhibited acceptable linearity (r>0.9966), intraassay precision ranged from 3.8 to 9.6%, interassay precision ranged from 3.0 to 17.0%, and accuracy was within 81.4-112.0%. This FMTAD derivatization method is considered very useful for determination of vitamin D analogs in ESI and applicable for biological samples.     

Development and validation of a liquid chromatography-tandem mass spectrometry for the determination of BPR0L075, a novel antimicrotuble agent, in rat plasma: application to a pharmacokinetic study

A rapid and sensitive liquid chromatography-tandem mass spectrometric method (LC-MS/MS) had been developed and validated to determine the concentrations of BPR0L075 in rat plasma. After a simple protein precipitation of plasma samples by acetonitrile, BPR0L075 was analyzed on a C(8) column at a flow rate of 0.5 mL/min. The mobile phase consisted of a mixture of 10 mM ammonium acetate containing 0.1% formic acid and acetonitrile (20:80, v/v). Both BPR0L075 (analyte) and the internal standard (BPR0L092) were determined using electro-spray ionization and the MS data acquisition was via multiple reactions monitoring (MRM) in positive scanning model. The MS/MS ion transitions monitored are m/z 342.2/195.2 and 312.5/165.2 for BPR0L075 and BPR0L092, respectively. The low limit of quantitation was 0.5 ng/mL. Each plasma sample was chromatographed within 5 min. The method was validated with respect to linearity, accuracy, precision, recovery, and stability. A good linear relationship was observed over the concentration range of 0.5-1000 ng/mL (r>0.9994). Absolute recoveries ranged from 63.45 to 68.34% in plasma at the concentrations of 2, 40, 400, and 800 ng/mL. The intra- and inter-day accuracy ranged from 92.04 to 111.80%. Intra- and inter-day relative standard deviations were 1.08-3.29% and 1.96-5.46%, respectively. This developed and validated assay method had been successfully applied to a pharmacokinetic study after intravenous injection of BPR0L075 in rats at a dose of 5mg/kg.