Development and validation of a HPLC–ES-MS/MS method for the determination of glucosamine in human synovial fluid

A new HPLC method for the determination of glucosamine (2-amino-2-deoxy-d-glucose) in human synovial fluid was developed and validated. Synovial fluid samples were analyzed after a simple protein precipitation step with trichloroacetic acid using a polymer-based amino column with a mobile phase composed of 10 mM ammonium acetate (pH 7.5)–acetonitrile (20:80, v/v) at 0.3 mL/min flow rate. d-[1-¹³C]glucosamine was used as internal standard. Selective detection was performed by tandem mass spectrometry with electrospray source, operating in positive ionization mode and in multiple reaction monitoring acquisition (m/z 180 → 72 and 181 → 73 for glucosamine and internal standard, respectively). The limit of quantification (injected volume = 3 μL) was 0.02 ng, corresponding to 10 ng/mL in synovial fluid. Calibration curves obtained using matrix-matched calibration standards and internal standard at 600 ng/mL were linear up to 2000 ng/mL. Precision values (%R.S.D.) were ≤14% in the entire analytical range. Accuracy (%bias) ranged from −11% to 10%. The recoveries measured at three concentration levels (50, 800, and 1500 ng/mL) were higher than 89%. The method was successfully applied to measure endogenous glucosamine levels in synovial fluid samples collected from patients with knee osteoarthritis and glucosamine levels after oral administration of glucosamine sulfate (DONA^®) at the dose of 1500 mg/day for 14 consecutive days (steady-state).     

LC-MS/MS method for simultaneous determination of viramidine and ribavirin levels in monkey red blood cells

A high performance liquid chromatography-tandem mass spectrometry (LC-MS/MS) method has been developed for the simultaneous determinations of total viramidine (viramidine, viramidine monophosphate, viramidine diphosphate, and viramidine triphosphate) and total ribavirin (ribavirin, ribavirin monophosphate, ribavirin diphosphate, and ribavirin triphosphate) in monkey red blood cells (RBC). The method involves the addition of internal standards and perchloric acid, conversion of viramidine or ribavirin phosphorylated metabolites to viramidine or ribavirin, purification with an aminopropyl (NH(2)) solid phase extraction (SPE) cartridge, and LC-MS/MS analysis. The MS/MS is selected to monitor m/z 245-->113, 250-->113, 244-->112, and 249-->112 for ribavirin, [(13)C]ribavirin, viramidine, and [(13)C]viramidine, respectively, using positive electrospray ionization. The calibration curves are linear over a concentration range of 100-10,000 ng/mL (0.412-41.2 microM) with a lower limit of quantification (LLOQ) of 100 ng/mL for both compounds. Mean inter-assay recoveries for ribavirin are 101%, 98.9%, and 96.0%, with coefficient of variance (%CV) values between 1.95 and 4.50% for 100, 1000, and 10,000 ng/mL quality control (QC) samples, respectively. Mean inter-assay recoveries for viramidine are 96.3%, 101%, and 102%, with coefficient of variation (%CV) values between 3.61 and 7.22%, for 100, 1000, and 10,000 ng/mL QC samples, respectively. Over-curve dilution QC at 400 microg/mL (1639 microM) for both viramidine and ribavirin are used to ensure the dilution accuracy (25 X dilutions) for monkey samples. The method has been used to simultaneously determine the total concentrations of ribavirin and viramidine in monkey RBC following 5, 15, and 36 weeks dosing of viramidine or ribavirin (60 mg/kg). The concentrations of total ribavirin following ribavirin dosing are 1242 microM at week 5, 1257 microM at week 15, and 1146 microM at week 36. The concentrations of total ribavirin following viramidine dosing are 634 microM at week 5, 716 microM at week 15, and 683 microM at week 36. Only small amounts of viramidine are detected in RBC following viramidine dosing, 7.80 microM at week 5, 6.63 microM at week 15, and 10.4 microM at week 36. The results suggest that ribavirin levels in RBC were at steady state at week 5 of ribavirin or viramidine dosing. At steady state, ribavirin levels in RBC are approximately 2x after ribavirin dosing than viramidine dosing. The relatively small percentage of viramidine in RBC suggests that viramidine either poorly penetrated into RBC or was extensively converted to ribavirin following entry into RBC.     

Comparative pharmacokinetics of trandolapril,its active metabolite,and verapamil in human plasma of Egyptian population using HPLC–MS/MS

Trandolapril and verapamil are commonly used antihypertensive drugs. However, there is a lack of available data on the change of their pharmacokinetics in patients with liver or kidney impairment and hence the need for dose adjustment. In this article, a high performance liquid chromatography–tandem mass spectrometry (HPLC–MS/MS) method was developed and validated for the monitoring of trandolapril, its active metabolite trandolaprilat, and verapamil in human plasma of patients with renal impairment and/or liver insufficiency. The chromatographic separation was achieved on a Gemini C₁₈ reversed phase column using a gradient elution mode with a run time of 10 minutes. The mobile phase consisted of a mixture of methanol and 2% acetic acid. The electrospray ionization MS/MS analysis was performed in multiple reaction monitoring (MRM) mode. The assay was validated as per Food and Drug Administration (FDA) guidelines for bioanalytical method validation and proved to be suitable for the determination of therapeutic drug levels in plasma. The inter‐group changes in pharmacokinetic data were compared to that of healthy volunteers. The comparison showed a significant difference in the pharmacokinetic parameters between the studied groups. The presented results exhibit the benefits of the proposed assay as a validated analytical tool for the continuous drug monitoring.     

Development of a rapid LC-MS/MS method for ribavirin determination in rat brain

A rapid and specific liquid chromatography tandem mass spectrometry (LC-MS/MS) method for the determination of ribavirin (RBV) in rat brain was developed. Sample preparation required only two centrifuge steps before LC-MS/MS analysis and the chromatographic separation was achieved in isocratic conditions using an Atlantis T3 column with a nearly totally aqueous (95%) mobile phase. The method showed a good linearity over a concentration range of 5-1000ppb and satisfactory results in terms of accuracy.     

Metabolite fingerprinting of Camptotheca acuminata and the HPLC–ESI-MS/MS analysis of camptothecin and related alkaloids

The major phytochemical constituents, namely, alkaloids, flavonoids and ellagic acid derivatives, of leaves of Camptotheca acuminata were identified using high performance liquid chromatography (HPLC) coupled with electrospray mass spectrometry (ESI-MS) in extracts of plants cultivated in Italy and collected at different growth stages. Alkaloids related to camptothecin were identified and quantified by HPLC coupled with ESI-tandem mass spectrometry (MS/MS) employing, respectively, an ion trap and a triple quadrupole mass analyser. The fragmentation patterns of alkaloids related to camptothecin were analysed and a specific Multiple Reaction Monitoring HPLC–MS/MS method was developed for the quantitative determination of these constituents. The described method provides high sensitivity and specificity for the characterisation and quantitative determination of the alkaloids in C. acuminata.     

Development of a new GC–MS/MS method for the determination of metformin in human hair

Diabetes mellitus is one of the most important public health challenges. Metformin (1,1‐dimethylbiguanide) represents the “gold standard” for the treatment of diabetes mellitus type 2. Despite its important role in reducing mortality and morbidity in the diabetic population, metformin is associated with an increased risk of stroke. To document exposure to a drug, hair is considered to be the specimen of choice to complement blood and urine, since it provides historical detail of a subject's chronic exposure to drug(s). Measuring hair concentration of metformin can be important for forensic toxicologists investigating criminal poisoning or Munchausen's syndrome by proxy. In clinical toxicology, drug monitoring using hair to document metformin observance has not yet been described. To document the interest of hair analysis for metformin, the authors have developed and validated a method using a gas‐chromatography tandem mass spectrometry system and applied it to authentic hair obtained from 9 diabetic patients under daily treatment. The validation procedure demonstrated a LOD an LOQ of 1 and 100 pg/mg, respectively and acceptable linearity, repeatability and reproducibility. The hair of the 9 patients tested positive in the low ng/mg range with concentrations ranging from 0.3 to 3.8 ng/mg. It seems obvious, in comparison with other drugs, that metformin is badly incorporated into hair, as the daily dosage varied from 1 to 3 g. Although limited in the number of subjects, the study allowed to postulate a possible correlation between daily dose and concentration in dark hair, while for light hair no correlation was found.     

Rapid UPLC–MS/MS method for the determination of ketoprofen in human dermal microdialysis samples

Dermal microdialysis (DMD) is a technique capable of determining the percutaneous penetration of drugs from topical formulations intended for local and/or regional activity. Typically, the concentrations of drug collected in dialysates are very low, generally in the ng/ml or even pg/ml range. An additional challenge is the very low volume of sample collected at each collection time and which can range from 1 to 30 μl only. Hence the objective was to develop and validate a rapid, accurate, precise, reproducible and highly sensitive LC–MS/MS method for the quantitative analysis of ketoprofen (KET) in dialystes following application of a topical gel product to the skin of human subjects. UPLC–MS/MS was used and KET was separated on an Acquity™ UPLC BEH C₁₈ column (100 mm × 2.1 mm i.d., 1.7 μm) and analysed in negative-ion (NI) electrospray ionisation (ESI) mode. The mobile phase (MP) consisted of acetonitrile:methanol:water (60:20:20, v/v/v) under isocratic conditions at a flow rate of 0.3 ml/min. Samples were extracted using ethyl acetate with ibuprofen (IBU) as internal standard (IS) and the organic solvent was then evaporated to dryness and the residue re-constituted in methanol. 5 μl samples were injected and analysis was performed at ambient temperature 22 ± 0.5 °C. KET and IBU eluted at 1.07 and 1.49 min, respectively. KET and IBU responses were optimised at the transitions 253.00 > 209.00 and 205.00 > 161.00, respectively. Calibration curves were linear over the range 0.5–500 ng/ml with correlation coefficients > 0.999. The accuracy and precision of the method were found to be between 99.97% and 104.67% (R.S.D. < 2%) and the mean recovery of KET from normal saline was 88.03 ± 0.3% (R.S.D. < 2.20%). The LLOQ and LOD values were found to be 0.5 and 0.1 ng/ml respectively whereas the ULOD was set at 500 ng/ml. The method was successfully applied to determine the bioavailability of KET following application of topical KET gel, Fastum^® gel, to the skin of human volunteers.     

Development and validation of LC–MS/MS method for the determination of cyproheptadine in several pharmaceutical syrup formulations

A rapid and sensitive liquid chromatographic–tandem mass spectrometric (LC–MS/MS) method was developed and validated for the qualitative and quantitative assay of cyproheptadine (CP) in pharmaceutical samples. Diphenylpyraline hydrochloride (DPP) was used as an internal standard (IS). Two multiple reaction-monitoring (MRM) transitions for each analyte were observed: 288.1/96.1 and 288.1/191.2 for CP and 282.1/167.2 and 282.1/116.3 for DPP. The retention time of the drug was 7.29 min. The analytical method was successfully validated for linearity (1–100 ng/ml), intra-day precision, inter-day precision, and accuracy. The limit of detection (LOD) and limit of quantification (LOQ) were 0.86 and 0.98 ng/ml, respectively. The proposed method was applied to analyse the cyproheptadine content from seven different syrup formulations.     

An LC–MS/MS method for determination of bioactive components of liquorice and Semen Strychni in rat plasma: Application to a pharmacokinetics study

Semen Strychni is known for its treatment of rheumatic arthritis with a low therapeutic index. Liquorice contributes a lot in herb detoxification according to the traditional Chinese medicine theory. A simple, rapid, and sensitive liquid chromatography–mass spectrometric method (LC–MS) was developed and validated for simultaneous determination of main bioactive ingredients in liquorice and Semen Strychni in rat plasma. Using moclobemide and cyproterone acetate as the internal standards, the analytes were pretreated via protein precipitation with methanol. An Ultimate AQ‐C18 column (3.0 μm, 3.0 × 100 mm) was employed for chromatographic separation, combining with gradient elution. The mobile phase consisted of 0.07% formic acid and 0.12% ammonium acetate in aqueous phase (A) and acetonitrile in organic phase (B). The elution program was as follows: 0–0.5 min, 20% B; 0.5–1 min, 20–60% B; 1–7 min, 60–85% B; and 7–7.5 min, returned to 20% B, then continued to 12 min. Selected reaction monitoring was performed in both positive and negative ESI. Positive mode was adopted for detection of strychnine, brucine, and moclobemide, while negative mode was used for glycyrrhizic acid, glycyrrhetinic acid, liquiritigenin, isoliquiritigenin, liquiritin, and cyproterone acetate. The method was validated for specificity, linearity, matrix effect, recovery, precision, accuracy, and stability. The results show that this method is sensitive, accurate and robust for biological matrix analysis. Moreover, the proposed method was applied to a pharmacokinetic study in Sprague–Dawley rats for investigating the mechanism of which liquorice detoxifies Semen Strychni.     

Development,validation, and application of an LC–MS/MS method for mitragynine and 7‐hydroxymitragynine analysis in hair

The entire scalp hair of a self‐declared Kratom consumer of 3 grams per day was acquired during an ethical committee approved study. As no values of the concentration in hair of the two Kratom alkaloids mitragynine or 7‐hydroxymitragynine were found in the literature, an already established method for the analysis of benzodiazepines/z‐substances was extended for the detection of mitragynine and 7‐hydroxymitragynine with LC–MS/MS, and successfully validated. The limits of detection and quantification for mitragynine were 2 pg/mg and 4 pg/mg, respectively. Those of 7‐hydroxymitragynine were 20 pg/mg and 30 pg/mg, respectively. The method was applied to the entire scalp hair, divided in 91 regions, of the study participant. A narrow mitragynine concentration distribution with values between 1054 pg/mg and 2244 ng/mg (mean 1517 ng/mg) and no clear scalp region associated distribution pattern was obtained. 7‐Hydroxymitragynine was not detected in any hair sample. After validation, the method was established as routine and subsequently 300 samples (mainly abstinence controls for drugs of abuse) were analyzed, allowing the investigation of the prevalence of Kratom consumption in our population. None of the analyzed routine hair samples were positive for mitragynine or 7‐hydroxymitragynine, providing no evidence that Kratom consumption is prevalent in the investigated population.     

Determination of S-propargyl-cysteine in rat plasma by mixed-mode reversed-phase and cation-exchange HPLC-MS/MS method and its application to pharmacokinetic studies

A simple, fast and sensitive mixed-mode reversed-phase and cation-exchange HPLC-MS/MS method for the quantification of S-propargyl-cysteine (SPRC), a novel cardioprotective agent, has been developed and validated for preclinical studies. Chromatographic separation of SPRC and its internal standard (IS) was performed using a commercial analytical column which contained both C18 bonded silica particles and sulfonic acid cation-exchange particles. The optimized mobile phase was composed of acetonitrile/ammonium acetate buffer (10mM, pH 4): 30/70 (v/v). Quantification was conducted by multiple reaction monitoring (MRM) of the transitions of m/z 160.0 → 143.0 for SPRC and 178.1 → 160.9 for S-butyl-cysteine (IS). The assay utilized methanol to achieve a simple and fast deproteinization. The lower limit of quantification (LLOQ) was 0.6 μg/mL (diluted with 50-fold of methanol) using 20 μL rat plasma. The assay was linear over a range from 0.6 to 159 μg/mL, with intra- and inter-batch accuracy (as relative error) less than ± 5% and precision (as relative standard deviation) less than 10%. Using the validated assay, the pharmacokinetic properties of SPRC in rats were investigated. SPRC exhibits linear pharmacokinetics after oral or intravenous administration in rats. The bioavailability after oral administration at 25, 75, and 225 mg/kg was 96.6%, 97.0%, and 94.7%, respectively.     

Sensitive and robust UPLC–MS/MS method to determine the gender-dependent pharmacokinetics in rats of emodin and its glucuronide

In this study, a sensitive and robust ultraperformance liquid chromatography–tandem mass spectrometry (UPLC–MS/MS) method was developed, validated, and applied to determine gender-dependent pharmacokinetics of total emodin (aglycone + glucuronide) in male and female Sprague–Dawley rats. The lower limit of quantification for emodin and emodin glucuronide in rat plasma was 39 and 78 ng/ml, with signal-to-noise ratio of ≥10. Precision and accuracy studies showed emodin and emodin glucuronide plasma concentrations well within the 10% range in all studies. Plasma recovery of emodin and emodin glucuronide was always above 86% for low (emodin: 39 ng/ml; glucuronide: 78 ng/ml), 92% for medium (625 ng/ml), and 97% for high (10 000 ng/ml) concentrations. Furthermore, emodin showed more than 95% plasma stability under short-term and long-term storage conditions, as well as after three freeze–thaw cycles in the experiments. The developed and validated analytical method was successfully applied to study the gender-dependent 10-fold higher oral bioavailability of total emodin in male than female rats. The oral bioavailability of emodin and emodin glucuronide was also measured separately and showed a statistically significant gender difference in oral bioavailability of emodin and emodin glucuronide in rats.     

A direct determination of thymidine triphosphate concentrations without dephosphorylation in peripheral blood mononuclear cells by LC/MS/MS

A rapid, sensitive and specific analytical method with minimal sample preparation for the measurement of thymidine triphosphate (TTP) in peripheral blood mononuclear cells (PBMC) by LC/MS/MS has been developed. PBMC were separated from whole blood or buffy coat. The analyte and internal standard were extracted from PBMC with 70% methanol (pH 7.2). These extracts after centrifugation were directly injected onto LC/MS/MS without need for any further sample preparation. The calibration curve was linear over the range 0.8–800 ng/ml. Mean inter- and intra-assay coefficients of variation (CVs) over the range of the standard curve were less than 10%. The overall recovery of TTP was 103.5%.     

Development of a multi‐residue high‐throughput UHPLC–MS/MS method for routine monitoring of SARM compounds in equine and bovine blood

Selective androgen receptor modulators (SARMs) are a group of anabolic enhancer drugs posing threats to the integrity of animal sports and the safety of animal‐derived foods. The current research describes for the first time the development of a semi‐quantitative assay for the monitoring of SARM family compounds in blood and establishes the relative stability of these analytes under various storage conditions prior to analysis. The presented screening method validation was performed in line with current EU legislation for the inspection of livestock and produce of animal origin, with detection capability (CCβ) values determined at 0.5 ng/mL (Ly2452473), 1 ng/mL (AC‐262536 and PF‐06260414), 2 ng/mL (bicalutamide, GLPG0492, LGD‐2226, ostarine, S‐1, S‐6, and S‐23), and 5 ng/mL (andarine, BMS‐564929, LGD‐4033, RAD140, and S‐9), respectively. The applicability of the developed assay was demonstrated through the analysis of blood samples from racehorses and cattle. The developed method presents a high‐throughput cost‐effective tool for the routine screening for a range of SARM compounds in sport and livestock animals.     

Evaluation of UV–HPLC and mass spectrometry methods for specific activity determination

The specific activity (SA) values determined using two different methods were compared for a set of tritium‐labeled and carbon‐14‐labeled compounds. The methods employed were as follows: (a) liquid chromatography/mass spectrometry (LC/MS) isotopic peak intensity distribution, and (b) determination of the tracer mass concentration using ultraviolet–high‐performance liquid chromatography analysis coupled with the radioactive solution concentration measured by liquid scintillation counting. In general, at lower SA, the accuracy and or precision of the LC/MS‐determined SA value decreased significantly. Because of this decrease in accuracy, a rough guideline of ~10% of the theoretical maximum SA is recommended as the lower cutoff for MS‐based SA measurements. If the tracer contains heteroatoms that possess significant percentages of heavy isotopes at natural abundance (e.g. Cl and Br), then the MS‐based SA cutoff recommendation is approximately 25–30% of the fully labeled compound in the tracer mixture. Additionally, IsoPat² was found to be the preferred calculation method for LC/MS‐based SA determination because SA values via this program were more consistent with those obtained by ultraviolet concentration calibration with solution count.     

Determination of GHB in human hair by HPLC‐MS/MS: Development and validation of a method and application to a study group and three possible single exposure cases

Gamma‐hydroxybutyrate (GHB) over the last two decades has generated increased notoriety as a euphoric and disinhibiting drug of abuse in cases of drug‐related sexual assault and for this reason it is considered a ‘date rape’ drug. The first aim of this paper was to develop and fully validate a method for the detection of GHB in human hair by high performance liquid chromatography‐tandem mass spectrometry (HPLC‐MS/MS) after liquid‐liquid extraction (LLE). The second aim was the application of the method to hair samples of 30 GHB‐free users in order to determine the basal level. The results obtained showed no significant differences in endogenous concentrations (p = 0.556) between hair samples of the three groups (black, blonde, and dyed hair) and the age and sex of the subjects did not affect the endogenous levels. Another 12 healthy volunteers, with no previous history of GHB use, were selected and a single dose (25 mg/Kg) was orally administered to all of them and hair samples were collected before the administration of the single dose and other two samples were collected one month and two months later, respectively. The segmental analysis of the latter two samples allowed us to calculate two ratios: 4.45:1 (95% C.I. 3.52–5.63) and 3.35:1 (95% C.I. 2.14–5.18), respectively, which can be recommended as reasonable values for a positive identification of GHB intake. Finally the method was applied to three real cases where a GHB single exposure probably occurred. Copyright © 2014 John Wiley & Sons, Ltd.     

A sensitive LC–MS/MS method to quantify loureirin B in rat plasma with application to preclinical pharmacokinetic studies

A novel method for the quantification of loureirin B in rat plasma using high-performance liquid chromatography/tandem mass spectrometry (LC–MS/MS) was developed. Loureirin B and internal standard (buspirone) were extracted by liquid–liquid extraction and separated on a Agilent XDB C₁₈ column (50 mm × 4.6 mm, 5 μm). As mobile phase a binary mixture of methanol (containing 0.1% formic acid)–water (containing 0.1% formic acid) was delivered by a Shimadzu LC-20AD pump in gradient mode at a flow rate of 0.4 ml/min in a run time of 5.0 min. The detector was a Q-trap™ mass spectrometer with an electrospray ionization (ESI) interface operating in the multiple reaction monitoring (MRM) mode. The calibration curve of loureirin B in plasma showed good linearity over the concentration range of 0.08–100 ng/ml. The limit of detection and limit of quantification were 0.03 ng/ml and 0.08 ng/ml, respectively. Intra- and inter-day precisions (as relative standard deviation) in all samples were both within 15%. The validated method was successfully applied to a preliminary pharmacokinetic study of loureirin B in rats. After oral administration of 16 g/kg longxuejie to rats, the main pharmacokinetic parameters t_max, C_max, t_1/2, K_e and AUC_0–T were 0.8 h, 7.99 μg/l, 1.94 l h, 0.365/h, and 22.21 μg h/l, respectively.