Dilution Integrity Research Articles

Simultaneous determination of nimesulide and its four possible metabolites in human plasma by LC-MS/MS and its application in a study of pharmacokinetics.

In this study, it was the first time that we simultaneously quantified nimesulide and its possible metabolites M1, M2, M3 and M4 by employing liquid chromatography-tandem mass spectrometry (LC-MS/MS). Nimesulide-d5 was used as internal standard (IS) for validation. Analytes and IS were recovered from human plasma by protein precipitation with acetonitrile. Prepared plasma samples were analyzed under the same LC-MS/MS conditions, and chromatographic separation was realized by using an Ultimate C18 column, with run time being 5min for each sample. Our results showed that various analytes within their concentration ranges could be quantified accurately by using the method. Mean intra- and inter-day accuracies ranged from -4.8% to 4.8% (RE), and intra- and inter-assay precision ≤6.2% (RSD). The following parameters were validated: specificity, recovery, matrix effects, dilution integrity, carry-over, sample stability under a variety of storage and handling conditions (room temperature, freezer, freeze-thaw and post-preparative) and stock solution stability. Pharmacokinetics of nimesulide and its metabolites were calculated based on the analysis of samples collected from twelve Chinese healthy volunteers after single oral dose of 100mg nimesulide tablets. By applying the pharmacokinetic determination into human samples, we preliminarily detected a new metabolite of nimesulide (M4*), and the concentration of M4* was relatively higher in plasma. Furthermore, we predicted part of conceivable metabolism pathway in plasma of after oral administration of 100mg nimesulide tablets. This research provided an experimental basis for further studies on metabolic activation and biotransformation of nimesulide, and for more comprehensive conjecture of its metabolic pathways.

Hair testing in clinical setting: Simultaneous determination of 50 psychoactive drugs and metabolites in headache patients by LC tandem MS

Headache patients suffering from recurrent attacks are a population at risk of overuse and abuse of analgesic medications. Associated with triptans, the first-line drugs recommended for the acute treatment, these patients usually take other medications such as opioids analgesics for the attack treatment, antidepressants and antiepileptics for prophylaxis treatment and benzodiazepines, non-benzodiazepine hypnotics and antipsychotics for the treatment of comorbidities. Regular and frequent use of triptans, like of any other symptomatic analgesic, can cause chronic headache and medication-overuse headache (MOH). In these circumstances, a detoxification treatment is necessary and therefore the monitoring and follow-up of the patients are crucial to the success of the treatment. In the present study, a LC tandem MS method has been developed for the identification of 50 psychoactive drugs in human hair, including triptans, benzodiazepines and metabolites, analgesics, antiepileptic, antidepressants and metabolites, a non-benzodiazepine hypnotic (z-drug), antipsychotics and metabolites. Hair samples were decontaminated, pulverized and incubated overnight in methanol; the extracts were then purified by a new and rapid QuEChERS procedure and analyzed by LC–MS/MS under gradient elution with positive ionization MRM mode. The procedure was fully validated in terms of selectivity, linearity, limit of detection and lower limit of quantitation, precision and accuracy, carry-over, matrix effect, recovery and dilution integrity. The validated procedure has been applied to 234 real hair samples collected from headache patients with known type and dosage of the taken drugs; the obtained data could be of interest to evaluate the xenobiotic concentrations in patients with known therapy.

Simultaneous determination of parecoxib sodium and its active metabolite valdecoxib in rat plasma by UPLC-MS/MS and its application to a pharmacokinetic study after intravenous and intramuscular administration.

In this study, we developed and validated a new, rapid, specific and sensitive ultra-performance liquid chromatography-tandem mass spectrometric (UPLC-MS/MS) method to simultaneously determine parecoxib sodium (PX) and its active metabolite, valdecoxib (VX), in rat plasma. Plasma samples were prepared by plasma protein precipitation combined with a liquid-liquid extraction method. The separation was carried out on a Kinetex C18 column (2.1mm×50mm, 2.6μm) with a gradient elution using methanol (A) and a 2mM ammonium acetate aqueous solution (B). The analysis was performed in less than 3min with a flow rate of 0.2mL/min. Ketoprofen was used as an internal standard (IS). Mass spectrometric detection was conducted with a triple quadrupole detector equipped with electrospray ionization in the negative ion mode (ESI(-)) using multiple reaction monitoring (MRM). The calibration curves were linear over the concentration ranges of 5-4000ng/mL for PX and 5-2000ng/mL for VX with all correlation coefficients greater than 0.998. The intra- and inter-day relative standard deviations (RSD) for both analytes were within 15% and the accuracy was within 85-115% at all quality control levels. The mean extraction recoveries for all analytes obtained from three concentrations of QC plasma samples were more than 89.0% efficient. Selectivity, matrix effect, dilution integrity and stability were also validated. The method was successfully used to investigate the pharmacokinetics of PX and VX in rat plasma after intravenous and intramuscular administration of PX.

Analysis of bacopaside I in biomatrices using liquid chromatography-tandem mass spectrometry: Pharmacokinetics and brain distribution in Swiss-albino mice

Bacopaside I (BP-I) is the major pseudojujubogenin glycoside of Bacopa monniera (BM) extract which has been widely used as a nerve tonic to improve the memory and intellect of human beings from ancient times. A selective and sensitive liquid chromatography–tandem mass spectrometry (LC–MS/MS) method for the quantification of BP-I in mouse plasma and brain homogenate has been developed and validated. All biosamples were processed by liquid-liquid extraction and chromatographed on C18- reversed phase column using mobile phase consisting of ammonium acetate (10mM, pH 4) – acetonitrile (10:90, v/v) at a flow rate of 0.5mL/min. The detection was performed in negative electrospray ionization mode and the precursor/product ion transitions of BP-I and internal standard (IS) hydrochlorothiazide were quantified in multiple reaction monitoring (MRM) using QTRAP-5500 MS/MS. The linearity was established over the concentration range of 0.5–2000ng/mL (r2>0.990), with lower limit of quantification (LLOQ) of 0.5ng/mL in both plasma and brain matrix. Within- and between-run precision and accuracy were well within the acceptable limits of variation. Consistent and reproducible recovery (>70%) was obtained with insignificant matrix effect for BP-I and IS. The method fulfilled US Food and Drug Administration (USFDA) guidelines for bioanalytical method validation in terms of selectivity, sensitivity, linearity, accuracy, precision, matrix effect, dilution integrity, carry-over effect and stability. Further, the method was successfully applied to execute the plasma pharmacokinetics and brain distribution of BP-I in Swiss-albino mice following intravenous administration at a dose of 5mg/kg.

Rapid, Selective and Rugged LCMS/MS Method Development and Validation for the Estimation of Clarithromycin in Dog Plasma

A new, simple, fast, sensitive and selective high performance liquid chromatography (HPLC) coupled with electrospray ionization tandem mass spectrometric method (LC-MS/MS) was developed and validated for the determination of clarithromycin in beagle dog plasma using imipramine as internal standard. The analyte was chromatographed under isocratic conditions on inertsil ODS 3 V (250X4.6 mm) 5μ column using mobile phase of 0.1% Formic acid: Methanol (15:85% v/v). The column temperature and flow rate was 40 ± 5°C and 1 ml/min respectively. The retention time for clarithromycin is within 1.52 min. Analysis was performed in the negative ionization mode using multiple reaction monitoring of the ion transitions at m/z 748.37/157.83 for clarithromycin and m/z 280.98/85.94 for internal standard, respectively. The validation was carried out over 9.922 - 4037.459 ng/ml of clarithromycin with the correlation coefficient of ≥ 0.9950. Method was validated for linearity, precision, accuracy, specificity, sensitivity, matrix effect, dilution integrity, ruggedness, reinjection reproducibility and stability. Clarithromycin was stable in stock solutions and in dog plasma samples under representative storage conditions. The present developed and validated method could be useful for the estimation of clarithromycin in beagle dog plasma.

Development and validation of a liquid chromatography-tandem mass spectrometric assay for quantitative analyses of triptans in hair.

Triptans are specific drugs widely used for acute treatment of migraine, being selective 5HT1B/1D receptor agonists. A proper assumption of triptans is very important for an effective treatment; nevertheless patients often underuse, misuse, overuse or use triptans inconsistently, i.e., not following the prescribed therapy. Drug analysis in hair can represent a powerful tool for monitoring the compliance of the patient to the therapy, since it can greatly increase the time-window of detection compared to analyses in biological fluids, such as plasma or urine. In the present study, a liquid chromatography-tandem mass spectrometric (LC-MS/MS) method has been developed and validated for the quantitative analysis in human hair of five triptans commonly prescribed in Italy: almotriptan (AL), eletriptan (EP), rizatriptan (RIZ), sumatriptan (SUM) and zolmitriptan (ZP). Hair samples were decontaminated and incubated overnight in diluted hydrochloric acid; the extracts were purified by mixed-mode SPE cartridges and analyzed by LC-MS/MS under gradient elution in positive multiple reaction monitoring (MRM) mode. The procedure was fully validated in terms of selectivity, linearity, limit of detection (LOD) and lower limit of quantitation (LLOQ), accuracy, precision, carry-over, recovery, matrix effect and dilution integrity. The method was linear in the range 10-1000pg/mg hair, with R(2) values of at least 0.990; the validated LLOQ values were in the range 5-7pg/mg hair. The method offered satisfactory precision (RSD <10%), accuracy (90-110%) and recovery (>85%) values. The validated procedure was applied on 147 authentic hair samples from subjects being treated in the Headache Centre of Modena University Hospital in order to verify the possibility of monitoring the corresponding hair levels for the taken triptans.

Application of a novel liquid chromatography/tandem mass spectrometry method for the determination of antazoline in human plasma: Result of ELEPHANT-I [ELEctrophysiological, pharmacokinetic and hemodynamic effects of PHenazolinum (ANTazoline mesylate)] human pharmacokinetic study

Antazoline is a first-generation antihistaminic agent with antiarrhythmic quinidine-like properties. In some countries, it is widely used for termination of cardiac arrhythmias, especially atrial fibrillation (AF). However, no human pharmacokinetic studies have been conducted with intravenous antazoline. The aim of our study was to develop and validate a novel liquid chromatography/tandem mass spectrometry (LC–MS/MS) method for the determination of antazoline in human plasma: the ELEPHANT-I [ELEctrophysiological, pharmacokinetic and hemodynamic effects of PHenazolinum (ANTazoline mesylate)] human pharmacokinetic study. Antazoline was extracted from plasma using liquid–liquid extraction. The concentration of the analyte was measured by LC–MS/MS with xylometazoline as an internal standard. The method was validated for linearity, precision, accuracy, stability (freeze/thaw stability, stability in autosampler, short and long term stability), dilution integrity and matrix effect. The analyzed validation criteria were fulfilled. The method was applied to a pharmacokinetic study involving 10 healthy volunteers. Following a single intravenous dose of antazoline mesylate (100mg), the plasma concentration profile showed a relative fast elimination with a terminal elimination half-life of 2.29h. A relatively high volume of distribution was observed (Vss=315L). The values of mean residence time (MRT∞), area under the curve (AUC∞) and clearance were 3.45h, 0.91mghL−1 and 80.5Lh−1, respectively. One volunteer showed significant differences in pharmacokinetic parameters. In conclusion, the proposed new LC–MS/MS method was successfully used for the first time for the determination of antazoline in human plasma.

Development of a rapid column-switching LC-MS/MS method for the quantification of THCCOOH and THCCOOH-glucuronide in whole blood for assessing cannabis consumption frequency.

The concentration of 11-nor-9-carboxy-Δ(9)-tetrahydrocannabinol (THCCOOH) in whole blood is used as a parameter for assessing the consumption behavior of cannabis consumers. The blood level of THCCOOH-glucuronide might provide additional information about the frequency of cannabis use. To verify this assumption, a column-switching liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the rapid and direct quantification of free and glucuronidated THCCOOH in human whole blood was newly developed. The method comprised protein precipitation, followed by injection of the processed sample onto a trapping column and subsequent gradient elution to an analytical column for separation and detection. The total LC run time was 4.5min. Detection of the analytes was accomplished by electrospray ionization in positive ion mode and selected reaction monitoring using a triple-stage quadrupole mass spectrometer. The method was fully validated by evaluating the following parameters: linearity, lower limit of quantification, accuracy and imprecision, selectivity, extraction efficiency, matrix effect, carry-over, dilution integrity, analyte stability, and re-injection reproducibility. All acceptance criteria were analyzed and the predefined criteria met. Linearity ranged from 5.0 to 500μg/L for both analytes. The method was successfully applied to whole blood samples from a large collective of cannabis consumers, demonstrating its applicability in the forensic field.

Determination of phenobarbital in hair matrix by liquid phase microextraction (LPME) and gas chromatography–mass spectrometry (GC–MS)

A method for identification and quantification of phenobarbital in hair samples by liquid phase microextraction (LPME) and gas chromatography–mass spectrometry (GC–MS) has been presented. Drug-free hair specimens were collected and separated in 50mg aliquots. Each aliquot was washed with 2.0mL of dichloromethane for 15min at 37°C. Standards and deuterated internal standards for calibration and quality control samples were added to the washed hair aliquot and the sample was submitted to complete digestion with sodium hydroxide (NaOH) 1.0mol/L for 15min at 70°C. The dissolved sample was submitted to LPME. After extraction, the residue was derivatized with tetramethylammonium hydroxide (TMAH) and analyzed by GC–MS. The limit of detection (LOD) was 0.1ng/mg and the limit of quantification (LOQ) was 0.25ng/mg. The calibration curve was linear over a concentration range of 0.25ng/mg to 10ng/mg (r2>0.99). The intra- and inter-assay precisions, given by RSD, were less than 6% for phenobarbital. Fortified samples of secobarbital and pentobarbital were also submitted to the validated method. The method was successfully applied to hair samples collected from three volunteers who reported regular use of phenobarbital (clinical treatment). The concentrations found were 9.5, 15.1 and 16.3ng/mg of phenobarbital. To contemplate the concentrations found, dilution integrity tests were also validated. The LPME and GC–MS method showed to be suitable for the detection of phenobarbital in hair samples and can be promptly used for different purposes whenever required.

Development and validation of a LC-MS/MS method for quantitative analysis of uraemic toxins p-cresol sulphate and indoxyl sulphate in saliva

p-Cresol sulphate (pCS) and indoxyl sulphate (IS) are uraemic toxins, the concentration of which in serum correlate with the stage of renal failure. The aim of this study was to develop and validate a high-performance liquid chromatography–tandem mass spectrometry (LC-MS/MS) method for the analysis of pCS and IS in saliva. This is the first time, to our knowledge, that such a method has been developed using saliva. Unstimulated, fasting saliva was collected from healthy volunteers in the morning and pooled for validation assay. The method was validated for linearity, precision, accuracy, stability (freeze/thaw stability, stability in autosampler, short- and long-term stability, stock solution stability), dilution integrity and matrix effect. The analysed validation criteria were fulfilled. No influence of salivary flow (pCS: p=0.678; IS: p=0.238) nor type of swab in the Salivette device was detected. Finally, using the novel validated method, the saliva samples of healthy people (n=70) of various ages were analysed. We observed a tendency for an increase of concentration of toxins in saliva in the elderly. This could be a result of age-related diseases, e.g., diabetes and kidney function decline. We can conclude that the novel LC-MS/MS method can be used for the determination of pCS and IS in human saliva. The results encourage the validation of saliva as a clinical sample for monitoring toxin levels in organisms.

A liquid chromatography-tandem mass spectrometry method for the quantitation of actarit in rabbit plasma: application to pharmacokinetics and metabolic stability.

Actarit (ATR), 4-acetylaminophenylacetic acid is an orally effective disease-modifying anti-rheumatic drug widely prescribed for the treatment of rheumatoid arthritis. The present study demonstrates the first report on a selective and sensitive liquid chromatography-tandem mass spectrometry method for the quantification of ATR in rabbit plasma using p-coumaric acid as an internal standard (IS). Following liquid-liquid extraction, chromatographic separation of the reconstituted samples was achieved isocratically on a Syncronis-C18 column with a mobile phase consisting of aqueous ammonium acetate (10 mM, pH 4)- methanol and acetonitrile mixture (8 : 92, v/v) at a flow rate of 0.6 ml/min. ATR and IS were detected using electrospray ionization operated in negative multiple reaction monitoring mode. The calibration curve was linear (r(2) ≥ 0.990) over the concentration range of 1-4000 ng/ml with a lower limit of quantitation of 1 ng/ml. The mean extraction recovery of ATR and IS from rabbit plasma was greater than 85%. The method complied well with US Food and Drug Administration guidelines for selectivity, sensitivity, accuracy, precision, matrix effect, dilution integrity, carry-over effect and stability. The method was successfully applied to in vitro metabolic stability (using rabbit liver microsomes) and in vivo pharmacokinetic study after oral administration of ATR at a dose of 10 mg/kg in New Zealand rabbits. Copyright © 2015 John Wiley & Sons, Ltd.

Quantitative analysis of pasireotide (SOM230), a cyclic peptide, in monkey plasma using liquid chromatography in combination with tandem mass spectrometry.

A novel liquid chromatographic method with tandem mass spectrometric detection (LC-MS/MS) for the determination of Pasireotide (SOM230) was developed and validated with a dynamic range of 0.5-250ng/ml using 50μl of monkey plasma. SOM230 and the internal standard, [M+6]SOM230, were extracted from monkey plasma via μElution SPE. The acidified sample matrix was loaded onto the preconditioned Waters SPE plate for further processing. The analyte was eluted from the SPE plate using freshly prepared elution solvent, which was followed by dilution and LC-MS/MS analysis. By eliminating a step of evaporation of the SPE eluent, instead, injecting the eluent after a simple dilution, compound loss due to non-specific binding to the 96-well materials was prevented. Furthermore, freshly prepared elution solution was found a key to optimal extraction recovery of the analyte from monkey plasma. The optimal chromatographic separation was achieved on an Atlantis dC18 (50×2.1mm, 5μm particle size) column using gradient elution with a total analysis cycle time approximately 4min per injection. The mobile phases were water containing 0.5% acetic acid and 0.05% trifluoroacetic acid (TFA) (mobile phase A) and acetonitrile containing 0.5% acetic acid and 0.05% TFA (mobile phase B). The incorporation of TFA (0.05%, v/v) and acetic acid (0.5%, v/v) into the mobile phases was accompanied by the improved chromatography and minimized carryover due to the HPLC column. The current method was validated for specificity, sensitivity, matrix effect, recovery, linearity, accuracy and precision, dilution integrity, batch size and stability. The accuracy and precision for the LLOQs (0.5ng/ml) were within ±5.6% bias and ≤7.8% CV, respectively. From the intra-day and inter-day evaluations, the precision of the other QC samples (1.5, 7.5, 75 and 190ng/ml) ranged from 2.7 to 4.9% CV and the accuracy (% bias) from -1.3 to 7.3%, respectively. Additional assessment of incurred sample reanalysis (ISR) was conducted to demonstrate the ruggedness and robustness of the assay method. The validated method was successfully implemented to support a toxicity study in monkeys administered with 5 and 30mg of SOM230 in a single intramuscular injection of a long acting release (LAR) formulation.

Quantitation of the enantiomers of tramadol and its three main metabolites in human whole blood using LC–MS/MS

The analgesic drug tramadol and its metabolites are chiral compounds, with the (+)- and (−)-enantiomers showing different pharmacological and toxicological effects. This novel enantioselective method, based on LC–MS/MS in reversed phase mode, enabled measurement of the parent compound and its three main metabolites O-desmethyltramadol, N-desmethyltramadol and N,O-didesmethyltramadol simultaneously. Whole blood samples of 0.5g were fortified with internal standards (tramadol-13C-D3 and O-desmethyl-cis-tramadol-D6) and extracted under basic conditions (pH 11) by liquid–liquid extraction. Chromatography was performed on a chiral alpha-1-acid glycoprotein (AGP) column preceded by an AGP guard column. The mobile phase consisted of 0.8% acetonitrile and 99.2% ammonium acetate (20mM, pH 7.2). A post-column infusion with 0.05% formic acid in acetonitrile was used to enhance sensitivity. Quantitation as well as enantiomeric ratio measurements were covered by quality controls. Validation parameters for all eight enantiomers included selectivity (high), matrix effects (no ion suppression/enhancement), calibration model (linear, weight 1/X2, in the range of 0.25–250ng/g), limit of quantitation (0.125–0.50ng/g), repeatability (2–6%) and intermediate precision (2–7%), accuracy (83–114%), dilution integrity (98–115%), carry over (not exceeding 0.07%) and stability (stable in blood and extract). The method was applied to blood samples from a healthy volunteer administrated a single 100mg dose and to a case sample concerning an impaired driver, which confirmed its applicability in human pharmacokinetic studies as well as in toxicological and forensic investigations.

Rapid, Selective, and Rugged Method Development and Validation of Atorvastatin and its Active Metabolites, o-Hydroxy Atorvastatin and p-Hydroxy Atorvastatin, in Stabilized Plasma Using Liquid Chromatography Coupled with Tandem Mass Spectrometry

A simple, sensitive, selective, and rugged liquid chromatography coupled with mass spectrometry (LC-MS/MS) method for the quantification of atorvastatin and its active metabolites, p-hydroxy atorvastatin and o-hydroxy atorvastatin, in human plasma was developed and validated. Isocratic elution of atorvastatin and its metabolites were achieved in 4.8 min using Luna 5 μ, C18, 100 mm × 4.60 mm column having a mobile phase of acetonitrile: 0.20% formic acid (65:35% v/v). The flow rate was 0.60 mL/min at a column temperature of 40 ± 5°C. Electron spray ionization technique in negative mode was selected to improve the selectivity and sensitivity required for this application. The retention times of atorvastatin, p-hydroxy atorvastatin and o-hydroxy atorvastatin were 3.73, 2.34, and 3.33 min, respectively. The method was validated for linearity, precision, accuracy, specificity, sensitivity, matrix effect, dilution integrity, ruggedness, reinjection reproducibility, and stability. Calibration curves during the course of validation were found to be linear for atorvastatin, p-hydroxy atorvastatin, and o-hydroxy atorvastatin in the ranges of 204.554–202,809.991, 197.836–196,149.459, and 202.316–200,590.719 pg/mL with correlation coefficient ≥0.9969, 0.9958, and 0.9970, respectively, and by using a 1/x2 weighted least square regression analysis of standard plots associated with ten-point calibration standards. The precision and mean accuracy were within the acceptable limits.

Systematic evaluation of matrix effect and cross-talk-free method for simultaneous determination of zolmitriptan and N-desmethyl zolmitriptan in human plasma: a sensitive LC-MS/MS method validation and its application to a clinical pharmacokinetic study.

The objective of the present work was to carry out systematic evaluation to eliminate matrix effect owing to plasma phospholipids as observed during sample preparation and to develop a cross-talk-free sensitive, selective and rapid bioanalytical method for the simultaneous determination of zolmitriptan (ZT) and N-desmethyl zolmitriptan (DZT) in human plasma by liquid chromatography-tandem mass spectrometry using naratriptan as internal standard (IS). The analytes and IS were quantitatively extracted from 200 μL human plasma by solid phase extraction. No cross-talk was found between ZT and DZT having identical product ions. Quantitation was performed on a triple quadrupole mass spectrometer employing electrospray ionization technique, operating in multiple reaction monitoring and positive ion mode. The total chromatographic run time was 2.5 min. The method was fully validated for sensitivity, selectivity, specificity, linearity, accuracy, precision, recovery, matrix effect, dilution integrity and stability studies. The method was validated over a dynamic concentration range of 0.1-15 ng/mL for ZT and DZT. The method was successfully applied to a bioequivalence study of 2.5 mg ZT tablet formulation in 18 healthy Indian male subjects under fasting conditions. Assay reproducibility was assessed by reanalysis of 62 incurred samples.

Rapid determination of benzodiazepines, zolpidem and their metabolites in urine using direct injection liquid chromatography–tandem mass spectrometry

Benzodiazepines and zolpidem are generally prescribed as sedative, hypnotics, anxiolytics or anticonvulsants. These drugs, however, are frequently misused in drug-facilitated crime. Therefore, a rapid and simple liquid chromatography–tandem mass spectrometric (LC–MS/MS) method was developed for identification and quantification of benzodiazepines, zolpidem and their metabolites in urine using deuterium labeled internal standards (IS). Urine samples (120μL) mixed with 80μL of the IS solution were centrifuged. An aliquot (5μL) of the sample solution was directly injected into the LC–MS/MS system for analysis. The mobile phases consisted of water and acetonitrile containing 2mM ammonium trifluoroacetate and 0.2% acetic acid. The analytical column was a Zorbax SB-C18 (100mm×2.1mm i.d., 3.5μm, Agilent). The separation and detection of 18 analytes were achieved within 10min. Calibration curves were linear over the concentration ranges of 0.5–20ng/mL (zolpidem), 1.0–40ng/mL (flurazepam and temazepam), 2.5–100ng/mL (7-aminoclonazepam, 1-hydroxymidazolam, midazolam, flunitrazepam and alprazolam), 5.0–200ng/mL (zolpidem phenyl-4-carboxylic acid, α-hydroxyalprazolam, oxazepam, nordiazepam, triazolam, diazepam and α-hydroxytriazolam), 10–400ng/mL (lorazepam and desalkylflurazepam) and 10–100ng/mL (N-desmethylflunitrazepam) with the coefficients of determination (r2) above 0.9971. The dilution integrity of the analytes was examined for supplementation of short linear range. Dilution precision and accuracy were tested using two, four and ten-folds dilutions and they ranged from 3.7 to 14.4% and −12.8 to 12.5%, respectively. The process efficiency for this method was 63.0–104.6%. Intra- and inter-day precisions were less than 11.8% and 9.1%, while intra- and inter-day accuracies were less than −10.0 to 8.2%, respectively. The lower limits of quantification were lower than 10ng/mL for each analyte. The applicability of the developed method was successfully verified with human urine samples from drug users (n=21). Direct urine sample injection and optimized mobile phases were introduced for simple sample preparation and high-sensitivity with the desired separation.

Development of an LC-MS/MS method for simultaneous determination of memantine and donepezil in rat plasma and its application to pharmacokinetic study.

Recently, a fixed dose combination (FDC) of memantine (MM) and donepezil (DPZ) has been approved for the treatment of Alzheimer's disease (AD). In the present work, a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the simultaneous determination of MM and DPZ was developed and validated in rat plasma over the linearity range of 0.2-400ng/mL using amantadine (AM) as an internal standard. Both the analytes and IS were extracted using one step liquid-liquid extraction procedure. The analytes were separated on C18 reversed phase column with mobile phase consisting of a mixture of methanol and 10mM ammonium acetate, pH 5 (92:8 v/v) at a flow rate of 0.7mL/min. The detection of the analytes was done on triple quadrupole mass spectrometer operated in positive electrospray ionization mode (ESI) and quantified using multiple reaction monitoring (MRM). The method was fully validated in terms of linearity, accuracy, precision, recovery, matrix effect, dilution integrity, carry-over effect and stability. The within- and between-run precisions were <10% and accuracy was all within ±10%. The mean recovery of MM and DPZ was found to be greater than 80%. The % RSD value at higher as well as lower concentration was well within the acceptable range (±15%) in all the stability experiments. The method was successfully applied to the oral pharmacokinetics and drug-drug interaction study of MM and DPZ in male Sprague Dawley (SD) rats.

Praziquantel efficacy against Brachylaima sp. metacercariae (Trematoda: Brachylaimidae) parasitizing the edible landsnail Cornu aspersum and its HPLC-MS/MS residue determination

Cornu aspersum is a terrestrial edible snail, often parasitized by Brachylaima (Trematoda) metacercariae. Ingestion of undercooked snails by humans allows metacercariae to develop to adult in the intestine causing brachylaimiasis (expected mortality rate 5–10%). The cosmopolitan character of the trematode and of its vector, enhanced in a future climate change scenario, and the absence of adequate sanitary controls of the snails in marketplaces clearly increase the risk of human brachylaimiasis. The treatment of farmed snails with praziquatel (PZQ) would be a tool to control this food-borne disease. The objectives of this study were: to report the prevalence of Brachylaima metacercariae parasitizing C. aspersum in marketplaces, to assess the efficacy and tolerance of PZQ in C. aspersum and to develop an HPLC-MS/MS analytical method to quantify PZQ residue in the edible snail (the acceptable daily intake of PZQ is 0.17 mg/kg bw in humans). Prevalence of parasitization of C. aspersum by Brachylaima metacercariae in public marketplaces reached the 80%. PZQ was orally administered to snails, mixed with the usual snail food. In dose determination assays three doses were individually tested (10 days): 1.2 mg PZQ/snail, 1.8 mg PZQ/snail (efficacy 97.3%, p < 0.001) and 2.4 mg PZQ/snail (efficacy 98.7%, p < 0.001). In dose confirmation tests (n = 200) the 2.4 mg PZQ/snail dose was group tested (10 snails/box, 7 days): 2 g of PZQ supplemented snail food were disposed daily in the treatment group boxes and 2 g of snail food (placebo) in the control boxes (efficacy 94.6%, p < 0.001; prevalence dropped from 68.7% in control group to 10.1% in treatment group, p < 0.001). Received dose was 220.2 mg PZQ/kg snail with shell. In the analytical method, linearity, lower limit of quantification (0.05 μg/ml), selectivity, carry over, accuracy, precision, dilution integrity, matrix effect and stability were tested. Sixty snails were treated (11 mg PZQ/g snail food) and analyzed. PZQ was detected and quantified (0.093 mg PZQ/g snail). PZQ treatment of C. aspersum is effective, well tolerated by the snail, affordable and easy to reproduce in snail farms.

Development and validation of a rapid ultra high performance liquid chromatography with tandem mass spectrometry method for the simultaneous determination of darunavir, ritonavir, and tenofovir in human plasma: Application to human pharmacokinetics

A sensitive ultra high performance liquid chromatography with tandem mass spectrometry method was developed for the simultaneous determination of darunavir, ritonavir and tenofovir in human plasma. Sample preparation involved a simple liquid-liquid extraction using 200 μL of human plasma extracted with methyl tert-butyl ether for three analytes and internal standard. The separation was accomplished on an Acquity UPLC BEH C18 (50 mm x 2.1 mm, 1.7 μm) analytical column using gradient elution of acetonitrile/methanol (80:20, v/v) and 5.0 mM ammonium acetate containing 0.01% formic acid at a flow rate of 0.4 mL/min. The linearity of the method ranged between 20.0 and 12 000 ng/mL for darunavir, 2.0 and 2280 ng/mL for ritonavir, and 14.0 and 1600 ng/mL for tenofovir using 200 μL of plasma. The method was completely validated for its selectivity, sensitivity, linearity, precision and accuracy, recovery, matrix effect, stability, and dilution integrity. The extraction recoveries were consistent and ranged between 79.91 and 90.04% for all three analytes and internal standard. The method exhibited good intra-day and inter-day precision between 1.78 and 6.27%. Finally the method was successfully applied for human pharmacokinetic study in eight healthy male volunteers after the oral administration of 600 mg darunavir along with 100 mg ritonavir and 100 mg tenofovir as boosters.

Quantification of lamotrigine in patient plasma using a fast liquid chromatography-tandem mass spectrometry method with backflush technology.

Recent concerns have been raised by neurologists and patients with epilepsy regarding the bioequivalence of generic lamotrigine to the brand Lamictal. Bioequivalence studies require the quantification of lamotrigine in human plasma, including in the presence of other drugs, for studies that will use patients with epilepsy rather than healthy volunteers. Lamotrigine was extracted from plasma through a simple protein precipitation and analyzed by fast liquid chromatography coupled to heated electrospray ionization with tandem mass spectrometric detection. A backflush step to remove interferent accumulation on column was included, and a stable isotope-labeled lamotrigine was used as an internal standard. The method was validated for accuracy, precision, interday and intraday coefficient of variation, specificity, lower limit of detection, lower limit of quantification, linearity, range, instrument precision, freeze-thaw, dilution integrity, and sample stability. Specificity evaluation included consideration of the impact of other antiepileptic drugs. The described method has a linear range of 8-10,000 ng/mL of lamotrigine (r = 0.9999) and a lower limit of detection of 1 ng/mL and a lower limit of quantification of 8 ng/mL. Intraday and interday reproducibility were less than 10.0% relative SD and 10.4% relative SD, respectively, and the percent recovery varied from 96.6% to 109.3% at various lamotrigine concentrations. A backflush step reduced matrix effects and no interference peak from plasma or other antiepileptic drugs were observed. A liquid chromatography-heated electrospray ionization-tandem mass spectrometry method including a backflush step was developed and validated to measure lamotrigine concentration in patient plasma. The method will be applied to bioequivalence studies that compare brand versus generic lamotrigine.