Amantadine hydrochloride monitoring by dried plasma spot technique: High‐performance liquid chromatography–tandem mass spectrometry based clinical assay

Amantadine plasma concentrations correlate well with desired therapeutic effects and adverse outcomes; information on amantadine exposure could be useful when multiple amantadine clearance pathways are impaired or non‐compliance is suspected. Micro‐sampling strategies, like dried plasma spot, would be particularly useful because ambulatory patients that do not attend a clinic can easily sample a few drops of blood by themselves at the required time of the dosing interval. We developed and validated a dried‐plasma‐spot‐based high performance liquid chromatography–tandem mass spectrometry assay to quantify amantadine. This assay met relevant validation requirements within a hematocrit range of 20–50% and was linear from 100 to 2000 ng/mL. Amantadine was stable in dried plasma spots for up to 21 days at room temperature, regardless of whether the dried plasma spot was protected from light or not. The correlation between paired dried and wet plasma concentrations was assessed in 52 patients. Deming regression coefficients between wet plasma and simultaneously pipetted dried plasma spots were used to predict plasma concentrations. Bland–Altman plots revealed a strong agreement between dried and wet plasma concentrations, supporting the clinical usefulness of dried plasma spots for amantadine monitoring with a self‐sampling strategy at a convenient time and place for the patient.     

Application of tail vein serial microsampling for plasma or dried plasma spots in toxicokinetic assessment in rats using acetaminophen as the model compound

In the current study, two groups of rats (five per group) were administered a single oral dose of 500 mg/kg acetaminophen. For toxicokinetic assessment, the Group 1 animals were bled via conventional sparse (two animals/time point) sublingual vein bleeding (~0.5 ml) with anesthesia, while the Group 2 animals were bled via serial tail vein microsampling (~0.075 ml) without anesthesia. All collected blood was processed for plasma. Each Group 2 plasma sample (~30 μl) was divided into ‘wet’ and ‘dried’ (dried plasma spots). All plasma samples were analyzed by LC–MS/MS for acetaminophen and its major metabolites acetaminophen glucuronide and acetaminophen sulfate. In addition, plasma and urine samples were collected for analysis of corticosterone and creatinine to assess stress levels. Comparable plasma exposure to acetaminophen and its two metabolites was observed in the plasma obtained via conventional sparse sublingual vein bleeding and serial tail vein microsampling and between the ‘wet’ and ‘dried’ plasma obtained by the latter. Furthermore, comparable corticosterone levels or corticosterone/creatinine ratios between the two groups suggested that serial microsampling without anesthesia did not increase the levels of stress as compared with conventional sampling with anesthesia, confirming the utility of microsampling for plasma or dried plasma spots in rodent toxicokinetic assessment.     

Volumetric Absorptive Microsampling of Blood for Untargeted Lipidomics

In the present, proof-of-concept paper, we explore the potential of one common solid support for blood microsampling (dried blood spot, DBS) and a device (volumetric absorptive microsampling, VAMS) developed for the untargeted lipidomic profiling of human whole blood, performed by high-resolution LC-MS/MS. Dried blood microsamples obtained by means of DBS and VAMS were extracted with different solvent compositions and compared with fluid blood to evaluate their efficiency in profiling the lipid chemical space in the most broad way. Although more effort is needed to better characterize this approach, our results indicate that VAMS is a viable option for untargeted studies and its use will bring all the corresponding known advantages in the field of lipidomics, such as haematocrit independence.     

Hybridation of different chiral separation techniques with ICP-MS detection for the separation and determination of selenomethionine enantiomers: chiral speciation of selenized yeast

Enantioseparation and determination of selenomethionine enantiomers in selenized yeast was investigated using chiral separation techniques based on different principles, coupled on-line to inductively coupled plasma mass spectrometry (ICP-MS) for selenium-specific detection. High performance liquid chromatography (HPLC) on a beta-cyclodestrin (beta-CD) column, cyclodextrin-modified micellar electrokinetic chromatography (CD-MEKC), gas chromatography (GC) on a Chirasil-L-Val column, and HPLC on a Chirobiotic T column have been investigated as the chiral separation techniques. For HPLC separation on the beta-CD column, and also for CD-MEKC, selenomethionine enantiomers were derivatized with NDA/CN(-). For chiral separation by GC, selenomethionine enantiomers were converted into their N-trifluoroacetyl (TFA)-O-alkyl esters. The developed hybridation methodologies are compared with respect to enantioselectivity, sensitivity and analysis time. The usefulness of the best-suited method [HPLC (Chirobiotic T)-ICP-MS] was demonstrated by its application to the successful chiral speciation of selenium and D-and L-selenomethionine content determination in selenized yeast.     

Multiplexed therapeutic drug monitoring of antipsychotics in dried plasma spots by LC‐MS/MS

In this work, a convenient method for the therapeutic monitoring of seven common antipsychotic drugs in “dried plasma spot” samples has been developed. It is based on the liquid chromatography tandem mass spectrometry technique, operating in multiple reaction monitoring mode, and a straightforward procedure for the simultaneous extraction of all antipsychotics in a single step, with high extraction yield. The method was fully validated with proper accuracy, precision, selectivity and sensitivity, for all the drugs. Limits of quantification were 0.12, 1.09, 1.46, 1.47, 5.70, 1.32, 1.33 µg/L for haloperidol, aripiprazole, olanzapine, quetiapine, clozapine, risperidone, and paliperidone, respectively. Accuracy, intra‐ and interday precision values were <10% for all drugs at all concentration levels examined. The method was tested in the analysis of 30 plasma samples from real patients for each drug. The proposed analytical approach, by combining practical and logistical advantages of microsampling with liquid chromatography tandem mass spectrometry analytical performance, could offer an ideal strategy for accurate and timely therapeutic drug monitoring of antipsychotic drugs in most clinical settings, even in remote centers and/or in out‐patient settings, bringing so many potential improvements in psychiatric patient care.     

Direct chiral resolution of cloquintocet-mexyl and its application to in vitro degradation combined with clodinafop-propargyl

A simple chiral high‐performance liquid chromatography (HPLC) method with ultraviolet (UV) detection was developed and validated for measuring Cloquintocet‐mexyl (ClM) enantiomers and clodinafop‐propargyl (CP) using cellulose tris‐(3,5‐dimethylphenylcarbamate) (CDMPC) as chiral stationary phase (CSP). The effects of mobile phase composition and column temperature on the ClM enantiomer separation were investigated. Good separation was achieved by using a mixture of n‐hexane and n‐propanol as mobile phase. Based on the chiral HPLC method, enantioselective quantitative determination analysis methods for this herbicide combined with CP in diluted plasma were developed and validated. The assay method was linear over a range of concentrations (0.5–100 µg/mL) in diluted plasma and the mean recovery was greater than 80% for both enantiomers and CP. The limits of quantification and detection for both ClM enantiomers and CP were 0.5 and 0.2 µg/mL, respectively. Intra‐ and interday relative standard deviations did not exceed 10% for three tested concentrations. The result suggested that the degradation of ClM enantiomers was stereoselective in rabbit plasma, and both rac‐ClM and CP degraded quickly in plasma, showing that the main existing forms with biological effect in animals are their metabolites. Copyright © 2011 John Wiley & Sons, Ltd.     

Determination of the R-(-) and S-(+) enantiomers of the monohydroxylated metabolite of oxcarbazepine in human plasma by enantioselective high-performance liquid chromatography.

An enantioselective liquid chromatographic assay for the simultaneous determination of the S-(+) and R-(-) enantiomers of the monohydroxylated metabolite of oxcarbazepine in human plasma is described. The metabolite is the active principle. The method is based on the extraction of plasma with diethyl ether-dichloromethane (2:1, v/v), separation of the organic phase, evaporation of the solvent and dissolution of the residue in the mobile phase. The two enantiomers were resolved on a Chiralcel OD (250 mm x 4.6 mm I.D.) high-performance liquid chromatographic column. The separation was achieved by isocratic elution with n-hexane-2-propanol (77:23, v/v). The flow-rate of the mobile phase was 1.0 ml/min and the two enantiomers were detected by ultraviolet absorbance at 210 nm. The analytical method is suitable for the quantitative and simultaneous determination of the two enantiomers in plasma at concentrations down to 0.4 mumol/l after administration of oxcarbazepine.     

Recent development trends for chiral stationary phases based on chitosan derivatives,cyclofructan derivatives and chiral porous materials in high performance liquid chromatography

The separation of enantiomers by chromatographic methods, such as gas chromatography, high‐performance liquid chromatography and capillary electrochromatography, has become an increasingly significant challenge over the past few decades due to the demand of pharmaceutical, agrochemical, and food analysis. Among these chromatographic resolution methods, high‐performance liquid chromatography based on chiral stationary phases has become the most popular and effective method used for the analytical and preparative separation of optically active compounds. This review mainly focuses on the recent development trends for novel chiral stationary phases based on chitosan derivatives, cyclofructan derivatives, and chiral porous materials that include metal‐organic frameworks and covalent organic frameworks in high‐performance liquid chromatography. The enantioseparation performance and chiral recognition mechanisms of these newly developed chiral selectors toward enantiomers are discussed in detail.     

Enantioselective analysis of bambuterol in human plasma using microwave‐assisted chiral derivatization coupled with ultra high performance liquid chromatography and tandem mass spectrometry

In this study, an enantioselective analytical method based on microwave‐assisted chiral derivatization coupled with ultra high performance liquid chromatography and tandem mass spectrometry was developed for the determination of bambuterol enantiomers in human plasma. The chiral derivatization reaction was greatly accelerated by microwave irradiation. Under the optimized conditions, both the derivatization time and separation time on column was only 3 min, and the lower limit of quantification was 2.5 pg/mL. The recoveries were in the range of 90.1–93.0% without significant matrix effect. Compared with the conventional heating chiral derivatization, microwave‐assisted chiral derivatization obtained higher chiral derivatization yields with much shorter time due to the effect of microwave irradiation. Furthermore, the racemization during the derivatization reaction was systematically investigated. The results showed the concentration of acetic acid and the reaction time had significant effects on the racemization, which could be well controlled during microwave‐assisted chiral derivatization for the short reaction time. Finally, this novel approach was demonstrated by determining bambuterol in human plasma of a clinical pharmacokinetic study in eight healthy volunteers. On the basis of the results, microwave‐assisted chiral derivatization coupled with ultra high performance liquid chromatography and tandem mass spectrometry as a simple and effective enantioselective analysis technique for the determination of chiral drugs in complex biological samples showed great promise.     

Quantification of vincristine and tariquidar by liquid chromatography-tandem mass spectrometry in mouse whole blood using volumetric absorptive microsampling for pharmacokinetic applications

A liquid chromatography-tandem mass spectrometry method was developed and validated for the simultaneous quantification of vincristine and tariquidar in 10 μL of mouse whole blood using volumetric absorptive microsampling devices. Samples were extracted from the devices and quantified against calibrators prepared in a human blood plasma matrix. Separation of vincristine and tariquidar was achieved using a Shimpack XR ODS III C18 stationary phase and H₂O and methanol mobile phase solvents containing 0.1% formic acid, running a gradient elution at a flow rate of 0.2 mL/min over 6.0 min. The method was linear up to 1200 ng/mL (R² > 0.99 for both analytes), with calibrator accuracy within ± 15% of the nominal concentrations and analyte coefficient of variance <15% for both vincristine and tariquidar. Pharmacokinetic assessment of both analytes was successfully applied in mice as both single-agent therapy and combination therapy over a 24-h period, and a 2.3-fold increase in vincristine drug exposure was observed in combination with tariquidar. This study validates the use of this approach for longitudinal analysis of drug exposure in animal studies.     

Enantiomeric Separation and Determination of R,S-Salsolinol by Capillary Electrophoresis

A new method for the quantitative determination of the enantiomers of salsolinol, an endogenous neurotoxin, was reported. Enantiomeric separation of salsolinol was obtained by capillary zone electrophoresis using hydroxypropyl-β-cyclodextrin （HP-β-CD） as chiral selector. Coupled with UV detection, this separation was applied to the determination of R,S-salsolinol in dried banana chips, and both R- and S-salsolinol were found at 40 ktg/g level.     

Determination of gemifloxacin on dried blood spots by hydrophilic interaction liquid chromatography with fluorescence detector: application to pharmacokinetics in rats

A highly selective, sensitive and rapid hydrophilic liquid interaction chromatographic method was developed and validated for determination of gemifloxacin on dried blood spots. The chromatographic separation was achieved on a reversed‐phase zwitterionic hydrophilic interaction liquid chromatographic ZIC®HILIC‐C₁₈ (4.6 × 100 mm; 5 µm) column using acetonitrile–10 mM ammonium acetate (pH 3.5; 80:20, v/v) as a mobile phase in an isocratic elution mode at a flow rate 0.6 mL/min at 27 °C. An on‐line fluorescence detector set at excitation and emission wavelengths of 269 and 393 nm, respectively was used for monitoring column eluents. Ciprofloxacin was used as an internal standard. The method was validated for accuracy, precision, linearity and selectivity by design of experiments following ICH guidelines. The assay exhibited a linear range of 25–5000 ng/mL for gemifloxacin on dried blood spots. The lower limit of detection was found to be 10 ng/mL. The intra‐ and inter‐assay coefficients of variation did not exceed 7.4% deviation of the nominal concentration. The recovery of GFX from dried blood spots was >95.0% and its stability was excellent with no evidence of degradation during sample processing for at least 3 months storage in a freezer at ?20 °C. Copyright © 2012 John Wiley & Sons, Ltd.     

Determination of lansoprazole enantiomers in dog plasma by column‐switching liquid chromatography with tandem mass spectrometry and its application to a preclinical pharmacokinetic study

Lansoprazole, a selective proton pump inhibitor, has a chiral benzimidazole sulfoxide structure and is used for the treatment of gastric acid hypersecretory related diseases. To investigate its stereoselective pharmacokinetics, a column‐switching liquid chromatography with tandem mass spectrometry method was developed for the determination of lansoprazole enantiomers in dog plasma using (+)‐pantoprazole as an internal standard. After a simple protein precipitation procedure with acetonitrile, matrix components left behind after sample preparation were further eliminated from the sample by reversed‐phase chromatography on a C₁₈ column. The fluent was fed to a chiral column for the separation of lansoprazole enantiomers. Baseline separation of lansoprazole enantiomers was achieved on a Chiralcel OZ‐RH column using acetonitrile/0.1% formic acid in water (35:65, v/v) as the mobile phase at 40°C. The linearity of the calibration curves ranged from 3 to 800 ng/mL for each enantiomer. Intra‐ and inter‐day precisions ranged from 2.1 to 7.3% with an accuracy of ±1.7% for (+)‐lansoprazole, and from 1.6 to 6.9% with an accuracy of ±3.5% for (–)‐lansoprazole, respectively. The validated method was successfully applied for the stereoselective pharmacokinetic study of lansoprazole in beagle dog after intravenous infusion.     

Liquid chromatographic separation of the enantiomers of metoprolol and its alpha-hydroxy metabolite on Chiralcel OD for determination in plasma and urine.

The two enantiomers of metoprolol and the four enantiomeric forms of alpha-hydroxymetoprolol were separated by liquid chromatography on a Chiralcel OD column containing a cellulose tris(3,5-dimethyl-phenylcarbamate) chiral stationary phase. The column efficiency was strongly dependent on the flow-rate and the enantioselectivity was influenced by temperature. Of utmost importance for the chiral separation was the water content of the mobile organic phase. The separation system was used for the separation and determination of the enantiomers in plasma and urine samples. The metoprolol enantiomers could be determined by fluorescence down to 10 nmol/l of each in plasma with a relative standard deviation of less than 15%.     

Determination of the enantiomeric composition of ibuprofen in human plasma by high-performance liquid chromatography

A normal-phase high-performance liquid chromatographic method, using a hexane-ethyl acetate solvent system, for the determination of the enantiomeric composition of ibuprofen in human plasma is described. The method is based on the resolution of the diastereoisomeric amides formed on reaction of the ibuprofen enantiomers with S-1-(naphthen-1-yl)ethylamine using p-chlorophenoxy-acetic acid as internal standard. The application of the method for the determination of the enantiomeric composition of ibuprofen in human plasma following the repeated oral administration of the drug to two volunteers is reported. The plasma concentrations of the S-(+) enantiomer were always greater than that of the R-(-), the ratio of the areas under the enantiomer plasma concentration-time curves (S/R) being 1.8 and 1.6.     

Chiral separation of the β2‐sympathomimetic fenoterol by HPLC and capillary zone electrophoresis for pharmacokinetic studies

The development of methods for the separation of the enantiomers of fenoterol by chiral HPLC and capillary zone electrophoresis (CZE) is described. For the HPLC separation precolumn fluorescence derivatization with naphthyl isocyanate was applied. The resulting urea derivatives were resolved on a cellulose tris(3,5‐dimethylphenylcarbamate)‐coated silica gel column employing a column switching procedure. Detection was carried out fluorimetrically with a detection limit in the low ng/mL range. The method was adapted to the determination of fenoterol enantiomers in rat heart perfusates using liquid–liquid extraction. As an alternative a CE method was used for the direct separation of fenoterol enantiomers comparing different cyclodextrin derivatives as chiral selectors. Copyright © 2010 John Wiley & Sons, Ltd.     

One‐step strategy for the synthesis of a derivatized cyclodextrin‐based monolithic column

Derivatized β‐cyclodextrin (β‐CD) functionalized monolithic columns were prepared by a “one‐step” strategy using click chemistry. First, the intended derivatized β‐CD monomers were synthesized by a click reaction between propargyl methacrylate and mono‐6‐azido‐β‐CD and then sulfonation or methylation was carried out. Finally, monolithic columns were prepared through a one‐step in situ copolymerization of the derivatized β‐CD monomer and ethylene glycol dimethacrylate. The sulfated β‐CD‐based monolith was successfully applied to the hydrophilic interaction liquid chromatography separation of nucleosides and small peptides, while the methylated β‐CD‐functionalized monolith was useful for the separation of nonpolar compounds and drug enantiomers in capillary reversed‐phase liquid chromatography. The structures of the monomers were characterized by Fourier transform infrared spectroscopy and mass spectrometry. The physicochemical properties and column performance of monoliths were evaluated by scanning electron microscopy and micro high performance liquid chromatography. This strategy has considerable prospects for the preparation of other derivatized CD‐functionalized methacrylate monoliths.     

Green high‐performance liquid chromatography enantioseparation of lansoprazole using a cellulose‐based chiral stationary phase under ethanol/water mode

A simple and environmentally friendly reversed‐phase high‐performance liquid chromatography method for the separation of the enantiomers of lansoprazole has been developed. The chromatographic resolution was carried out on the cellulose‐based Chiralpak IC‐3 chiral stationary phase using a green and low‐toxicity ethanol‐aqueous mode. The effects of water content in the mobile phase and column temperature on the retention of the enantiomers of lansoprazole and its chiral and achiral related substances have been carefully investigated. A mixed‐mode hydrophilic interaction liquid chromatography and reversed‐phase retention mechanism operating on the IC‐3 chiral stationary phase allowed us to achieve simultaneous enantioselective and chemoselective separations in water‐rich conditions. The enantiomers of lansoprazole were baseline resolved with a mobile phase consisting of ethanol/water 50:50 without any interference coming from chiral and achiral impurities within 10 min.     

Rapid determination of rifaximin on dried blood spots by LC‐ESI‐MS

The use of blood spot collection cards is a simple way to obtain specimens for therapeutic drug monitoring, assessing adherence to medications and preventing toxicity in a clinical setting. A high‐throughput liquid chromatography–electrospray ionization mass spectrometric (LC‐ESI‐MS) method for determination of rifaximin on dried blood spots (DBS) was developed and validated. It involves solvent extraction of a punch of DBS followed by reversed‐phase LC on a monolithic column consisting of a silica rod with bimodal pore structure and detection by ESI‐MS. Rifampicin was used as an internal standard (IS). The run time was within 5.0 min with a very low back‐pressure at a flow rate of 0.5 mL/min. The assay was linear from 0.1 to 10 ng/mL. The mean recovery was 98.42%. The developed method is very simple, rapid and useful for clinical applications. Copyright © 2011 John Wiley & Sons, Ltd.