Determination of 17α-dihydroequilenin in rat,rabbit and monkey plasma by high-performance liquid chromatography with fluorimetric detection

A high-performance liquid chromatographic (HPLC) method with fluorescence detection for the determination of total (unconjugated and conjugated) 71α-dihydroequilenin in male and female rat rabbit and male rhesus monkey plasma is described here. Plasma sample preparation involved hydrolysis with enzyme (Glusulase), addition of internal standard (14β-equilenin) and solvent extraction. The extracts were chromatographed on a C₆, 5-μm reversed-phase HPLC column and detection was accomplished with a fluorescence detector operated at an excitation wavelength of 210 nm and an emission wavelength of 370 nm. The assay was linear over a range of 2.5 to 100 ng/ml in male and female rat plasma, and 5 to 500 ng/ml in female rabbit and male and female monkey plasma. The method was specific, accurate and reproducible (percent differences <14.5; coefficients of variation <9.5%) in all matrices examined. The applicability of this method was successfully tested by quantifying total plasma concentrations of 17α-dihydroequilenin in ovariectomized female rats, ovariectomized female rabbits and a normal female rhesus monkey receiving 2.0, 8.3 and 0.1 mg/kg, respectively, of 17α-dihydroequilenin sulfate intragastrically.     

Simultaneous determination of paracetamol,pseudoephedrine, dextrophan and chlorpheniramine in human plasma by liquid chromatography–tandem mass spectrometry

For the first time, a highly sensitive and simple LC–MS/MS method after one-step precipitation was developed and validated for the simultaneous determination of paracetamol (PA), pseudoephedrine (PE), dextrophan (DT) and chlorpheniramine (CP) in human plasma using diphenhydramine as internal standard (IS). The analytes and IS were separated on a YMC-ODS-AQ C₁₈ Column (100 mm × 2.0 mm, 3 μm) by a gradient program with mobile phase consisting of 0.3% (v/v) acetic acid and methanol at a flow rate of 0.30 mL/min. Detection was performed on a triple quadrupole tandem mass spectrometer via electrospray ionization in the positive ion mode. The method was validated and linear over the concentration range of 10–5000 ng/mL for PA, 2–1000 ng/mL for PE, 0.05–25 ng/mL for DT and 0.1–50 ng/mL for CP. The accuracies as determined from quality control samples were in range of ?8.37% to 3.13% for all analytes. Intra-day and inter-day precision for all analytes were less than 11.54% and 14.35%, respectively. This validated method was successfully applied to a randomized, two-period cross-over bioequivalence study in 20 healthy Chinese volunteers receiving multicomponent formulations containing 325 mg of paracetamol, 30 mg of pseudoephedrine hydrochloride, 15 mg of dextromethorphan hydrobromide and 2 mg of chlorphenamine maleate.     

Simultaneous determination of triprolidine and pseudoephedrine in human plasma by liquid chromatography–ion trap mass spectrometry

A highly efficient, selective and specific method for simultaneous quantitation of triprolidine and pseudoephedrine in human plasma by liquid chromatography–ion trap-tandem mass spectrometry coupled with electro spray ionization (LC–ESI-ion trap-tandem MS) has been validated and successfully applied to a clinical pharmacokinetic study. Both targeted compounds together with the internal standard (gabapentin) were extracted from the plasma by direct protein precipitation. Chromatographic separation was achieved on a C₁₈ ACE^® column (50.0 mm × 2.1 mm, 5 μm, Advance Chromatography Technologies, Aberdeen, UK), using an isocratic mobile phase, consisting of water, methanol and formic acid (55:45:0.5, v/v/v), at a flow-rate of 0.3 mL/min. The transition monitored (positive mode) was m/z 279.1 → m/z 208.1 for triprolidine, m/z 165.9 → m/z 148.0 for pseudoephedrine and m/z 172.0 → m/z 154.0 for gabapentin (IS). This method had a chromatographic run time of 5.0 min and a linear calibration curves ranged from 0.2 to 20.0 ng/mL for triprolidine and 5.0–500.0 ng/mL for pseudoephedrine. The within- and between-batch accuracy and precision (expressed as coefficient of variation, %C.V.) evaluated at four quality control levels were within 94.3–106.3% and 1.0–9.6% respectively. The mean recoveries of triprolidine, pseudoephedrine and gabapentin were 93.6, 76.3 and 82.0% respectively. Stability of triprolidine and pseudoephedrine was assessed under different storage conditions. The validated method was successfully employed for the bioequivalence study of triprolidine and pseudoephedrine formulation in twenty six volunteers under fasting conditions.     

Simultaneous determination of clarithromycin,rifampicin and their main metabolites in human plasma by liquid chromatography–tandem mass spectrometry

The drug combination rifampicin and clarithromycin is used in regimens for infections caused by Mycobacteria. Rifampicin is a CYP3A4 inducer while clarithromycin is known to inhibit CYP3A4. During combined therapy rifampicin concentrations may increase and clarithromycin concentrations may decrease. Therefore a simple, rapid and easy method for the measurement of the blood concentrations of these drugs and their main metabolites (14-hydroxyclarithromycin and 25-desacetylrifampicin) is developed to evaluate the effect of the drug interaction. The method is based on the precipitation of proteins in human serum with precipitation reagent containing the internal standard (cyanoimipramine) and subsequently high-performance liquid chromatography (HPLC) analysis and tandem mass spectrometry (MS/MS) detection in an electron positive mode. The method validation included selectivity, linearity, accuracy, precision, dilution integrity, recovery and stability according to the “Guidance for Industry – Bioanalytical Method Validation” of the FDA. The calibration curves were linear in the range of 0.10–10.0 mg/L for clarithromycin and 14-hydroxyclarithromycin and 0.20–5.0 mg/L for rifampicin and 25-desacetylrifampicin, with within-run and between-run precisions (CVs) in the range of 0% to ?10%. The components in human plasma are stable after freeze–thaw (three cycles), in the autosampler (3 days), in the refrigerator (3 days) and at room temperature (clarithromycin and 14-hydroxyclarithromycin: 3 days; rifampicin and 25-desacetylrifampicin: 1 day). The developed rapid and fully validated liquid chromatography–tandem mass spectrometry (LC/MS/MS) method is suitable for the determination of clarithromycin, 14-hydroxyclarithromycin, rifampicin and 25-desacetylrifampicin in human plasma.     

Simultaneous and sensitive determination of xanthotoxin,psoralen, isoimpinellin and bergapten in rat plasma by liquid chromatography–electrospray ionization mass spectrometry

A sensitive, specific and rapid liquid chromatography–mass spectrometry (LC–MS) method has been developed and validated for the simultaneous determination of xanthotoxin (8-methoxypsoralen), psoralen, isoimpinellin (5,8-dimethoxypsoralen) and bergapten (5-methoxypsoralen) in rat plasma using pimpinellin as an internal standard (IS). The plasma samples were pretreated by protein precipitation with methanol and chromatographic separation was performed on a C₁₈ column with a mobile phase composed of 1 mmol ammonium acetate and methanol (30:70, v/v). The detection was accomplished by multiple-reaction monitoring (MRM) scanning via electrospray ionization (ESI) source operating in the positive ionization mode. The optimized mass transition ion-pairs (m/z) for quantitation were 217.1/202.1 for xanthotoxin, 187.1/131.1 for psoralen, 247.1/217.0 for isoimpinellin, 217.1/202.1 for bergapten, and 247.1/231.1 for IS. The total run time was 6 min between injections. The calibration curves were linear over the investigated concentration range with all correlation coefficients higher than 0.998. The lower limits of quantitation (LLOQ) of these analytes were less than 1.21 ng/ml. The intra- and inter-day RSD were no more than 9.7% and the relative errors were within the range of ?8.1% to 4.5%. The average extraction recoveries for all compounds were between 90.7% and 106.2%. The proposed method was further applied to the determination of actual plasma samples from rats after oral administration of Radix Glehniae extract.     

Simultaneous determination of isoniazid,rifampicin, levofloxacin in mouse tissues and plasma by high performance liquid chromatography–tandem mass spectrometry

A rapid and selective high performance liquid chromatography–tandem mass spectrometry (HPLC–MS/MS) method for simultaneous determination of isoniazid (INH), rifampicin (RFP) and levofloxacin (LVX) in mouse tissues and plasma has been developed and validated, using gatifloxacin as the internal standard (I.S.). The compounds and I.S. were extracted from tissue homogenate and plasma by a protein precipitation procedure with methanol. The HPLC separation of the analytes was performed on a Welch materials C4 column (250 mm × 4.6 mm, 5.0 μm, USA) at 25 °C, using a gradient elution program with the initial mobile phase constituting of 0.05% formic acid and methanol (93:7, v/v) at a flow-rate of 1.0 ml/min. For all the three analytes, the recoveries varied between 83.3% and 98.8% in tissues and between 75.5% and 90.8% in plasma, the accuracies ranged from 91.7% to 112.0% in tissues and from 94.6% to 108.8% in plasma, and the intra- and inter-day precisions were less than 13.3% in tissues and less than 8.2% in plsama. Calibration ranges for INH were 0.11–5.42 μg/g in tissues and 0.18–9.04 μg/ml in plasma, for RFP were 0.12–1200 μg/g in tissues and 4.0–200 μg/ml in plasma, and for LVX were 0.13–26.2 μg/g in tissues and 0.09–4.53 μg/ml in plasma. The lower limits of quantification (LLOQs) for INH, RFP and LVX in mouse tissues were 0.11, 0.12 and 0.13 μg/g and for those in mouse plasma were 18.1, 20.0 and 21.8 ng/ml, respectively. The limits of detection (LODs) for INH, RFP and LVX in mouse tissues were 0.04, 0.05 and 0.05 μg/g and for those in mouse plasma were 5.5, 6.0 and 6.6 ng/ml, respectively. The established method was successfully applied to simultaneous determination of isoniazid, rifampicin and levofloxacin in mouse plasma and different mouse tissues.     

Simultaneous determination of m-nisoldipine and its three metabolites in rat plasma by liquid chromatography–mass spectrometry

A simple, sensitive and selective liquid chromatography–tandem mass spectrometry (LC–MS/MS) method for the simultaneous determination of m-nisoldipine and its three metabolites in rat plasma has been developed using nitrendipine as an internal standard (IS). Following liquid–liquid extraction, the analytes were separated using an isocratic mobile phase on a reverse phase C₁₈ column and analyzed by MS in the multiple reaction monitoring (MRM) mode. To avoid contamination by residual sample in the injection syringe, a special injection protocol was developed. We found that m-nisoldipine, metabolite M1 and IS could be ionized under positive or negative electrospray ionization conditions, whereas metabolite M and M2 could only be ionized in the positive mode. The mass spectrometry fragmentation pathways for these analytes are analyzed and discussed herein. The total analysis time required less than 5 min per sample. We employed this method successfully to study the metabolism of m-nisoldipine when it was orally administered to rats at a dose of 9 mg/kg. Three metabolites of m-nisoldipine and an unknown compound of molecular weight 386 were found for the first time in rat plasma. The concentration of the potentially active metabolite was approximately equal to its parent compound concentration.     

Determination of terbutaline enantiomers in human urine by coupled achiral–chiral high-performance liquid chromatography with fluorescence detection

A coupled achiral–chiral high-performance liquid chromatographic system with fluorescence detection at excitation/emission wavelengths of 276/306 nm has been developed for the determination of the enantiomers of terbutaline, (S)-(+)-terbutaline and (R)-(−)-terbutaline in urine. Urine samples were prepared by solid-phase extraction with Sep-pak silica, followed by HPLC. The terbutaline was preseparated from the interfering components in urine on Phenomenex silica column and the terbutaline enantiomers and betaxolol were resolved and determined on a Sumichiral OA-4900 chiral stationary phase. The two columns were connected by a switching valve equipped with silica precolumn. The precolumn was used to concentrate the terbutaline in the eluent from the achiral column before back flushing onto the chiral phase. For each enantiomer the assay was linear between 1 and 250 ng/ml (R²=0.9999) and the detection limit was 0.3 ng/ml. The intra-day variation was between 4.6 and 11.6% in relation to the measured concentration and the inter-day variation was 4.3–11.0%. It has been applied to the determination of (S)-(+)-terbutaline and (R)-(−)-terbutaline in urine from a healthy volunteer dosed with racemic terbutaline sulfate.     

Simultaneous determination of the enantiomers of leucine and [2H7]leucine in plasma by capillary gas chromatography–mass spectrometry

A method for the stereoselective assay of d- and l-enantiomers of both leucine and [²H₇]leucine in rat plasma was developed using gas chromatography–mass spectrometry–selected-ion monitoring. dl-[²H₃]leucine was used as an internal standard. The method involved purification by cation-exchange chromatography using BondElut SCX cartridge and derivatization with hydrochloric acid in methanol to form methyl ester followed by subsequent chiral derivatization with (+)-α-methoxy-α-trifluoromethylphenylacetyl chloride to form diastereomeric amide. The derivatization made the separation of the leucine enantiomers possible with good gas chromatographic behavior. Quantitation was performed by selected-ion monitoring of the quasi-molecular ions of the diastereomers on the chemical ionization method. The sensitivity, specificity, accuracy and reproducibility of the method were demonstrated to be satisfactory for application to pharmacokinetic studies of leucine enantiomers.     

Simultaneous quantification of venlafaxine and O-desmethylvenlafaxine in human plasma by ultra performance liquid chromatography–tandem mass spectrometry and its application in a pharmacokinetic study

A rapid, selective and sensitive ultra performance liquid chromatography–tandem mass spectrometry (UPLC-MS/MS) method was developed to simultaneously determine venlafaxine (VEN) and O-desmethylvenlafaxine (ODV) in human plasma. Sample pretreatment involved a one-step extraction with diethyl ether of 0.5 mL plasma. The separation was carried out on an ACQUITY UPLC? BEH C₁₈ column with 10 mmol/L ammonium acetate and methanol as the mobile phase at a flow rate of 0.30 mL/min. The detection was performed on a triple–quadrupole tandem mass spectrometer by multiple reaction monitoring (MRM) mode via electrospray ionization (ESI) source. The linear calibration curves for VEN and ODV were both obtained in the concentration range of 0.200–200 ng/mL (r² ≥ 0.99) with the lower limit of quantification (LLOQ) of 0.200 ng/mL. The intra- and inter-day precision (relative standard deviation, R.S.D.) values were less than 13% and the accuracy (relative error, R.E.) was within ±5.3% and ±3.6% for VEN and ODV. The method herein described was superior to previous methods in sensitivity and sample throughput and successfully applied to clinical pharmacokinetic study of venlafaxine sustained-release capsule in healthy male volunteers after oral administration.     

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.     

Simultaneous HPLC–UV determination of rhein and aceclofenac in human plasma

Diacerein and aceclofenac are prescribed for reducing the symptoms associated with osteoarthritis. We present a simple HPLC method with UV detection for simultaneous determination of rhein (the immediate metabolite of diacerein) and aceclofenac from human plasma samples. Sample preparation was accomplished through liquid–liquid extraction with ethyl acetate and chromatographic separation was performed on a reversed-phase ODS column. Mobile phase consisted of a mixture of acetate buffer and acetonitrile run under gradient at flow rate of 1.0 ml/min. Wavelength was set at 258 nm. The method was validated for linearity, accuracy, precision and stability. The calibration was linear over the range of 0.1–7.0 μg/ml for rhein and 0.5–20 μg/ml for aceclofenac using 500 μl plasma samples. Extraction recoveries were 85% for rhein and 70% for aceclofenac. The method can easily be adopted for high-throughput clinical and pharmacokinetic studies of above two-drug fixed dose combination formulations.     

Trace quantification of 1-octacosanol and 1-triacontanol and their main metabolites in plasma by liquid–liquid extraction coupled with gas chromatography–mass spectrometry

A method for the simultaneous determination of 1-octacosanol and 1-triacontanol and their main metabolites in rat plasma was developed. The procedure involved ethanolic NaOH saponification of the sample, acidification, liquid–liquid extraction, and derivatization of the analytes to its trimethylsilylether/ester, followed analysis by gas chromatography–mass spectrometry (GC–MS) in selected ion monitoring (SIM) mode. Quantification was performed by the internal standard method using betulin. The method had a good linearity over the range 8.4–540 ng/ml (r ≥ 0.998) and showed an excellent intra-day (R.S.D. = 0.59–3.06%) and inter-day (R.S.D. = 2.99–5.22%) precision according to the acceptance criteria. The detection limits ranged between 1.32 and 3.47 ng/ml. The method was applied successfully to study the total plasmatic concentration of 1-octacosanol, octacosanoic acid, 1-triacontanol, and triacontanoic acid, after an oral dose of policosanols mixture, using plasma samples of 100 μl.     

Simultaneous determination of thiamphenicol,florfenicol and florfenicol amine in swine muscle by liquid chromatography–tandem mass spectrometry with immunoaffinity chromatography clean-up

A rapid and sensitive liquid chromatography–electrospray ionization-tandem mass spectrometry (LC–ESI-MS/MS) method to quantify thiamphenicol (TAP), florfenicol (FF), and florfenicol amine (FFA) in swine muscle is described. An immunoaffinity chromatography (IAC) column based on polyclonal antibodies and protein A-sepharose CL 4B was used to clean-up extracted samples. IAC optimized conditions were found that allowed the IAC to be reused for selective binding of TAP, FF, and FFA. The dynamic column capacity was more than 512 ng/mL of gel after being used for 15 cycles. From fortified swine muscle samples at levels of 0.4–50 ng/g, the average recoveries were 85.2–98.9% with intra- and inter-day variations less than 9.8% and 12.4%, respectively. The limit of quantitation ranged from 0.4 to 4.0 μg/kg.     

Determination of free and liposomal Amphotericin B in human plasma by liquid chromatography–mass spectroscopy with solid phase extraction and protein precipitation techniques

Amphotericin B is available in various drug delivery systems such as cholesteryl sulfate complex, as lipid complex, and as liposomal formulation. The separation and measurement of free drug (drug which is not bound with liposomal lipids) and liposomal drug (drug which is entrapped in liposomes) in the human plasma after injection of liposomal Amphotericin B is of prime importance due to toxicity concerns. A robust, specific and sensitive method has been developed to effectively separate and then quantify the free drug and liposomal drug, present in human plasma. This method utilizes solid phase extraction Oasis HLB cartridges, which retains the free drug and the liposomal Amphotericin B was eluted from the cartridge in first step. The eluted liposomal Amphotericin B was then extracted from lipids by protein precipitation method using 2% dimethylsulfoxide (DMSO) in acetonitrile. After separation and extraction, the quantification of free and liposomal fractions of Amphotericin B was performed by HPLC–MS–MS technique. The chromatographic separation was performed using Chromolith Performance RP 18e column. The mobile phase composed of 5 mM ammonium acetate, methanol and acetonitrile and a gradient elution program was used. The calibration curves were found to be linear for free Amphotericin B (0.25–15.0 μg/ml) and liposomal Amphotericin B (1.0–100.0 μg/ml). The recovery was about 96% for free Amphotericin B and about 92% for liposomal Amphotericin B. Recoveries were consistent over the linearity ranges defined. The intra-batch and inter-batch accuracy and precision fulfilled the international requirements. The stability of free and liposomal Amphotericin B was assessed under different storage conditions.     

Simultaneous determination of 6-gingerol, 8-gingerol, 10-gingerol and 6-shogaol in rat plasma by liquid chromatography–mass spectrometry: Application to pharmacokinetics

A rapid high-performance liquid chromatography–mass spectrometry (HPLC–MS) method was developed and validated for simultaneous quantification of 6-gingerol, 8-gingerol, 10-gingerol and 6-shogaol in rat plasma after oral administration of ginger oleoresin. Plasma samples extracted with a liquid–liquid extraction procedure were separated on an Agilent Zorbax StableBond-C₁₈ column (4.6 mm × 50 mm, 1.8 μm) and detected by MS with electrospray ionization interface in positive selective ion monitoring (SIM) mode. Calibration curves (1/x² weighted) offered satisfactory linearity (r² > 0.995) in a wide linear range (0.0104–13.0 μg/mL for 6-gingerol, 0.00357–4.46 μg/mL for 8-gingerol, 0.00920–11.5 μg/mL for 10-gingerol and 0.00738–9.22 μg/mL for 6-shogaol). The lower limit of quantification (LLOQ) was in a range of 3.57–10.4 ng/mL. The analytes and internal standard can be baseline separated within 6 min. Inter- and intra-day assay variation was less than 15%. This developed method was successfully applied to pharmacokinetic studies of ginger oleoresin after oral administration to rats. Glucuronide of 6-gingerol was determined after β-glucuronidase hydrolysis for more information, and the intestinal glucuronidation was further confirmed by comparison of plasma samples of hepatic portal vein and femoral vein.     

Quantitation of itraconazole in rat heparinized plasma by liquid chromatography–mass spectrometry

A liquid chromatographic–mass spectrometric (LC–MS) assay was developed and validated for the determination of itraconazole (ITZ) in rat heparinized plasma using reversed-phase HPLC combined with positive atmospheric pressure ionization (API) mass spectrometry. After protein precipitation of plasma samples (0.1 ml) with acetonitrile containing nefazodone as an internal standard (I.S.), a 50-μl aliquot of the supernatant was mixed with 100 μl of 10 mM ammonium formate (pH 4.0). An aliquot of 25 μl of the mixture was injected onto a BDS Hypersil C₁₈ column (50×2 mm; 3 μm) at a flow-rate of 0.3 ml/min. The mobile phase comprising of 10 mM ammonium formate (pH 4) and acetonitrile (60:40, v/v) was used in an isocratic condition, and ITZ was detected in single ion monitoring (SIM) mode. Standard curves were linear (r²≥0.994) over the concentration range of 4–1000 ng/ml. The mean predicted concentrations of the quality control (QC) samples deviated by less than 10% from the corresponding nominal values; the intra-assay and inter-assay precision of the assay were within 8% relative standard deviation. Both ITZ and I.S. were stable in the injection solvent at room temperature for at least 24 h. The extraction recovery of ITZ was 96%. The validated assay was applied to a pharmacokinetic study of ITZ in rats following administration of a single dose of itraconazole (15 mg/kg).     

Determination of deoxynivalenol in organic and conventional food and feed by sol–gel immunoaffinity chromatography and HPLC–UV detection

The paper describes the determination of deoxynivalenol (DON) in 55 wheat food and feed samples, 26 from conventional and 29 from organic production. Immunoaffinity columns prepared by entrapping anti-DON antibodies by the sol–gel method were used for sample clean-up. DON was quantified by high performance liquid chromatography (HPLC) and ultraviolet (UV) detection. In general, the incidence of DON contamination was rather low. In eight samples (14.5%) the DON concentration was above the LOQ (380 ng/g), in six samples (10.9%) DON was detected but could not be quantified (>LOD (200 ng/g), <LOQ). In seven conventional samples (two pasta, two cookie, two snack and one feed sample) but only in one organic sample (a snack) the DON concentration was >LOQ. The data indicate both a higher incidence of DON contamination and higher DON concentrations in food and feed samples from conventional than in those from organic production.     

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.