Internal Standard Solution Research Articles

Rapid liquid chromatography-electrospray tandem mass spectrometry method for serum free and total carnitine.

The measurement of serum free carnitine (SFC) is useful for the investigation and monitoring of patients suffering from inherited metabolic disorders of fatty acid oxidation. Two tandem mass spectrometry (MS/MS) methods (1)(2) have recently been published to circumvent technical problems in the radioenzymatic reference method and the routine spectrophotometric enzymatic method. The method described by Hardy et al. (1) requires butyl esterification of SFC before MS/MS analysis and suffers from inaccuracy attributable to partial hydrolysis of acylcarnitines during the derivatization procedure. The method of Stevens et al. (2) does not require derivatization, but samples are first applied to cotton fiber filter paper, and SFC is eluted with a methanol–water solution before MS/MS analysis. Although not discussed by Stevens et al. (2), the filter paper serves to reduce serum sample matrix interference, a well-known phenomenon in electrospray ionization MS. The presence of any nonvolatile solutes in the biological sample matrix changes the droplet solution properties during electrospray ionization, causing ion suppression (3). On the other hand, liquid chromatography (LC) conditions have also been used to reduce matrix interference (4). We present an alternative LC MS/MS method for SFC that does not require derivatization and has reduced matrix interference. To 20 μL of serum sample, we added 300 μL of an internal standard solution containing 3.3 μmol/L 2H3-l-carnitine in acetonitrile to precipitate the serum protein in an Eppendorf tube. The mixture was vortex-mixed and sonicated for 10 min to improve the precision of the extraction procedure before centrifugation (7000 g for 5 min). The supernatant was then transferred to a sample vial for LC MS/MS analysis. A series of aqueous calibrators, ranging from 0 to 100 …

Approaches to applying internal standards for the quantification of paper additives by Py-GC/MSD

In the present study, five different methods of internal standard (IS) application with different IS substances were used for the quantification of typical chemical additives in paper such as wet strength or sizing agents by the combination of analytical pyrolysis with gas chromatographic separation and mass selective detection (Py-GC/MSD). The advantages and disadvantages are described. One approach only followed variations of the response of the MSD. A further approach, the injection of an IS solution within the pyrolyzer interface, compensated for variations only in the GC/MSD part. In the other three approaches the IS followed the whole analytical pathway. When different IS applications in the same run were used, sources for variations could be identified. The best reproducibility with relative standard deviations (RSDs) of 4–8% for the paper additives was obtained by using degradation products from the cellulosic matrix as internal standards. The shortcoming of this approach is that the matrix has to be known. Solutions to solve this problem are demonstrated.

Rapid and sensitive quantitation of the antiproliferative agent mitoguazone in small volumes of plasma by high-performance liquid chromatography with ultraviolet detection

Mitoguazone is an antiproliferative agent used in chemotherapy. This study describes a simple and sensitive high-performance liquid chromatographic method for the determination of mitoguazone in 100 μl of plasma. Samples were deproteinized with 100 μl of a solution of internal standard (amiloride, 10 μg/ml) in acetonitrile. An aliquot of the supernatant was injected onto the column. HPLC separation was achieved on a silica column with the mobile phase of methanol–50 m M potassium phosphate buffer (pH 3)–triethylamine (80:20:0.3, v/v), at a flow-rate of 1 ml/min. The eluent was detected at 320 nm. The retention time was about 5.5 min for amiloride and 12 min for mitoguazone. No endogenous substances were found to interfere. Calibration curves were linear from 0.25 to 50 μg/ml. The absolute recoveries of mitoguazone and amiloride were both greater than 84%. The limit of quantitation was 0.25 μg/ml. The intra- and inter-day precision (expressed as RSD) was 5.8%, or less, and the accuracy was 94.7% of the nominal concentration. The method is suitable in pharmacokinetic investigation and monitoring mitoguazone concentration.

A high accuracy reference method for the determination of minor elements in steel by ICP-OES

An ICP-OES method is described for the high accuracy analysis of minor elements in steel (Mn, Mo, Cr and Ni) with full uncertainty evaluation of ∼1% at the 95% (k = 2) confidence interval. The technique is ideally suited to the production of reference materials and standards. Samples are analysed by the exact matching technique in which a single elemental standard is prepared by closely matching the concentrations of sample and standard so that the emission intensity ratio of sample to standard is ∼1. Sample and standard are doped with an internal standard solution of gold to correct for instrumental drift. Critically, the sample and standard are also matched with respect to acid strength and Fe, the dominant matrix element. The Fe matrix is matched to within 3%. Without Fe addition, significant biases in ICP performance are observed. The single point calibration technique is readily amenable to an estimation of a complete uncertainty budget, taking into account all of the uncertainty contributions that derive from the protocol. The combined uncertainty is dominated by repeatability of the method, linked to sample preparation, rather than instrumental effects.

Serum Acylcarnitines and Vitamin B12 Deficiency

Measurements of acylcarnitines in plasma have several diagnostic applications, and various tandem mass spectrometry methods are more specific and accurate than earlier methods (1)(2)(3)(4). Libert et al. (5) reported that propionylcarnitine (PrCRN) and carnitine (CRN) esters of 2-methyl-branched fatty acids with chain lengths of four to nine carbons were found in the urine of patients with propionic and methylmalonic acidemia. Chace et al. (6) reported results for a high-throughput method for PrCRN analysis from dried-blood spots for the diagnosis of propionic and methylmalonic acidemias in newborns. Increased concentrations of methylmalonic acid (MMA) in urine or serum are also regarded as reliable indicators of functional vitamin B12 deficiency (7)(8)(9). The relationship between the concentrations of MMA and CRN esters in patients with increased MMA attributable to vitamin B12 deficiency has not been established. Because mass spectrometric testing is useful in newborn screening (10), with analysis of acylcarnitines, and specifically propionylcarnitine, to identify patients with methylmalonic acidemia, we evaluated the utility of acylcarnitine analysis in patients with low vitamin B12 concentrations. MMA was analyzed on a tandem mass spectrometer as described previously (11). Acylcarnitines were analyzed as follows. Aliquots (150 μL) of calibrators, controls, and patient sera were transferred into microcentrifuge tubes. Acetonitrile was added to each tube to bring the total volume to 650 μL. The tubes were vortex-mixed vigorously for 2 min and centrifuged at 10 000 g for 2 min; 50 μL of the organic phase was then transferred into an autosampler vial containing 50 μL of internal standard (IS) solution, containing d9-CRN, …

Validation of a micellar electrokinetic capillary chromatography method for the determination of imidurea, methyl and propylparabens in a pharmaceutical ointment

The quantitative aspects of a micellar electrokinetic chromatographic (MEKC) method for the determination of three preservatives (imidazolidinyl urea, methyl and propylparabens) in a pharmaceutical ointment are presented. Separation was carried out in a 31.2 cm long (21 cm to the detection window) × 50 μm i.d. fused silica capillary at an electric field of 960 V/cm and 25 °C. The electrolyte was 10 mM sodium dihydrogenophosphate containing 40 mM sodium dodecylsulfate (SDS) and adjusted to pH 6.1. Sample preparation consisted of a simple dispersion of the ointment in an internal standard solution (ethylparaben in the electrolyte). A 3.5 nl volume of the sample solution was introduced hydrodynamically into the capillary. The compounds detected at 200 nm were separated within 1.6 min. The method was validated with respect to specificity, linearity, accuracy, repeatability according to the International Conference on Harmonisation guidelines. Calibration curves were found to be linear for each compound ( r 2>0.999). Recovery studies performed by spiking an ointment placebo with the preservatives between 50 and 150% of their target concentration on 3 different days gave satisfactory results (mean recovery at each of the five concentrations ranging from 99.23 to 101.40%). The repeatability of sample preparation ( n=6) was better than 2% for each of the preservatives.

Quantification of copper and zinc species fractions in legume seeds extracts by SEC/ICP-MS: validation and uncertainty estimation

Fractions of Cu and Zn species in legume samples (common white bean, pea, chick pea and lentil seeds and defatted soybean flour) were analysed by on-line hyphenation of size exclusion chromatography and inductively coupled plasma-mass spectrometry. Samples were extracted by 0.02 mol l −1 Tris–HCl buffer solution, pH 7.5. The extraction efficiency lay in the region 60–90 and 60–80% for Cu and Zn, respectively. Quantification of elements in the individual chromatographic fractions was carried out by isotope dilution (ID) and external calibration (EC) techniques. For ID analysis the chromatographic effluent was mixed with the flow of 65Cu and 68Zn isotope enriched solution and the isotope ratio values 63Cu/ 65Cu and ( 64Zn+ 66Zn)/ 68Zn were measured. In the case of EC technique calibration solutions of elements were injected to the flow of mobile phase by the second injector. Prior entering detector the effluent was mixed with the flow of internal standard solution (In, 50 μg l −1). Both methods have similar precision, however the behaviour of both studied elements was not the same. The chromatographic analysis itself was the main source of variability in the case of Cu. For Zn species analysis, the extraction process and the manipulation with the extract, played the significant role too. It was probably caused by lower stability of the present zinc chelates. The total amounts of Zn found in all chromatographic fractions represented 85–95% of Zn in sampled extract whereas those of Cu approached 100%. In case of small peaks the results of ID and EC were not the same. The EC results were lower then ID results. The great deal of results uncertainty accounts for the precision.

Determination of delavirdine in very small volumes of plasma by high-performance liquid chromatography with fluorescence detection

Delavirdine is a newly developed anti-HIV-1 drug for AIDS therapy. This study describes a sensitive high-performance liquid chromatographic method for the determination of delavirdine in 50 μl of plasma. Samples were deproteinized with 150 μl of a solution of internal standard (cisapride 10 μg/ml) in acetonitrile. An aliquot of the supernatant was injected onto the column. HPLC separation was achieved on a C 18 column with the mobile phase of acetonitrile-50 m M sodium dihydrogen phosphate (60:40, v/v) at a flow-rate of 1 ml/min. The eluants were measured by fluorescence detection with excitation at 295 nm and emission filtration at 425 nm. The retention time was about 5.3 min for delavirdine and 6.5 min for cisapride. The specificity was demonstrated, as there were no interferences from plasma samples of different batches in the regions of peak interest. Calibration curves were linear from 25 to 25000 ng/ml. The limit of quantitation was 25 ng/ml. The within- and between-day precision (C.V.) was 9.3%, or less, and the accuracy was within 9.2% of the nominal concentration. The small sample volume needed is especially advantageous for the application both in pharmacokinetic studies in HIV-infected adults and pediatric patients, and in small animals, where limited samples are available.

魚油中のドコサヘキサエン酸およびｎ‐３脂肪酸の高分解能プロトン核磁気共鳴スペクトロスコピーによる定量分析 日本油化学会ワーキンググループ（ＤＨＡ／ＮＭＲ小委員会）による合同研究

Seven Japanese laboratories provided with high resolution nuclear magnetic resonance (NMR) equipment undertook a collaborative study for determining docosahexaenoic acid (DHA) and n-3 fatty acids in fish oils with NMR. This study was organized by the DHA/NMR subcommittee of the Japan Oil Chemists’ Society and conducted as a part of an IUPAC collaborative study (Project 3/98 of the commission VI-6 on Oils, Fats, and Derivatives). At each laboratory, examination was made of six blind duplicate fish oils, as follows; refined tuna oil, two extracted tuna oils, extracted bonito oil, extracted salmon oil and extracted sardine oil 9 to 30 mol% DHA and 20 to 35 mol% n-3 fatty acids. To ensure maximum accuracy of determination, signal area measurement and addition of the internal standard solution were taken as critical control points. Repeatability coefficient of variation (CVr) ranged from 0.8 to 2.6% and reproducibility coefficient of variation (CVR) ranged from 1.4 to 3.3% for DHA weight concentration (mg/g); CVr from 0.4 to 2.2% and CVR from 0.7 to 3.1% for DHA (mol%); CVr from 0.2 to 1.1% and CVR from 1.1 to 2.2% for total n-3 fatty acids (mol%). The present method was thus shown to qualify for use as a tentative official method of the Japan Oil Chemists’ Society.

Use of online-dual-column extraction in conjunction with chiral liquid chromatography tandem mass spectrometry for determination of terbutaline enantiomers in human plasma.

An online sample extraction chiral bioanalytical method was developed and validated for the quantification of terbutaline, a beta2-selective adrenoceptor agonist, spiked into human plasma by using two extraction columns and a chiral stationary phase (CSP) in conjunction with liquid chromatography tandem mass spectrometry (LC-MS/MS). In this method, two Oasis HLB extraction columns were used in parallel for plasma sample purification and a Chirobiotic T CSP was used for enantiomeric separation. Atmospheric pressure chemical ionization MS/MS was employed in multiple reaction monitoring mode for the detection and quantification. Subsequent to the addition of an internal standard solution, the plasma samples were directly injected onto the system for extraction and analysis. This method allowed the use of one of the extraction columns for purification while the other was being equilibrated. Hence, the time required for reconditioning the extraction columns did not contribute to the total analysis time per sample, which resulted in a shorter run time and higher throughput. A lower limit of quantification of 1.0 ng/mL was achieved using only 50 microliter of human plasma. The method was validated with a dynamic range of 1.0-200 ng/mL. The intra- and interday precision was no more than 11% CV and the assay accuracy was between 94-106%.

Parallel extraction columns and parallel analytical columns coupled with liquid chromatography/tandem mass spectrometry for on-line simultaneous quantification of a drug candidate and its six metabolites in dog plasma.

A method with parallel extraction columns and parallel analytical columns (PEC-PAC) for on-line high-flow liquid chromatography/tandem mass spectrometry (LC/MS/MS) was developed and validated for simultaneous quantification of a drug candidate and its six metabolites in dog plasma. Two on-line extraction columns were used in parallel for sample extraction and two analytical columns were used in parallel for separation and analysis. The plasma samples, after addition of an internal standard solution, were directly injected onto the PEC-PAC system for purification and analysis. This method allowed the use of one of the extraction columns for analyte purification while the other was being equilibrated. Similarly, one of the analytical columns was employed to separate the analytes while the other was undergoing equilibration. Therefore, the time needed for re-conditioning both extraction and analytical columns was not added to the total analysis time, which resulted in a shorter run time and higher throughput. Moreover, the on-line column extraction LC/MS/MS method made it possible to extract and analyze all seven analytes simultaneously with good precision and accuracy despite their chemical class diversity that included primary, secondary and tertiary amines, an alcohol, an aldehyde and a carboxylic acid. The method was validated with the standard curve ranging from 5.00 to 5000 ng/mL. The intra- and inter-day precision was no more than 8% CV and the assay accuracy was between 95 and 107%.

Quantitative analysis of active compounds in pharmaceutical preparations by use of attenuated total-reflection Fourier transform mid-infrared spectrophotometry and the internal standard method.

A new method is presented for the quantitative analysis of compounds in pharmaceutical preparations Fourier transform (FT) mid-infrared (MIR) spectroscopy with an attenuated total reflection (ATR) module. Reduction of the quantity of overlapping absorption bands, by interaction of the compound of interest with an appropriate solvent, and the employment of an internal standard (IS), makes MIR suitable for quantitative analysis. Vigabatrin, as active compound in vigabatrin 100-mg capsules, was used as a model compound for the development of the method. Vigabatrin was extracted from the capsule content with water after addition of a sodium thiosulfate IS solution. The extract was concentrated by volume reduction and applied to the FTMIR-ATR module. Concentrations of unknown samples were calculated from the ratio of the vigabatrin band area (1321-1610 cm(-1)) and the IS band area (883-1215 cm(-1)) using a calibration standard. The ratio of the area of the vigabatrin peak to that of the IS was linear with the concentration in the range of interest (90-110 mg, in twofold; n=2). The accuracy of the method in this range was 99.7-100.5% (n=5) with a variability of 0.4-1.3% (n=5). The comparison of the presented method with an HPLC assay showed similar results; the analysis of five vigabatrin 100-mg capsules resulted in a mean concentration of 102 mg with a variation of 2% with both methods.

Sensitive determination of four general anaesthetics in human whole blood by capillary gas chromatography with cryogenic oven trapping

Four general anaesthetics, sevoflurane, isoflurane, enflurane and halothane, in human whole blood, have been found measurable with very high sensitivity by capillary gas chromatography–flame ionization detection (GC–FID) with cryogenic oven trapping upon injection of headspace (HS) vapor sample. To a 7-ml vial, containing 0.48 ml of distilled water and 20 μl of internal standard solution (5 μg), a 0.5-ml of whole blood sample spiked with or without anaesthetics, was added, and the mixture was heated at 55°C for 15 min. A measure of 10 ml HS vapor was injected into the GC in the splitless mode at −40°C oven temperature, which was programmed up to 250°C. All four peaks were clearly separated; no impurity peaks were found among their peaks. Their extraction efficiencies were about 10%. The calibration curves showed good linearity in the range of 0.5–20 μg/ml; their detection limits were 10–100 ng/ml, which are almost comparable to those by previous reports. The coefficients of intra-day and day-to-day variations were 6.5–9.8 and 7.3–17.2%, respectively. Isoflurane or enflurane was also measured from whole blood samples in which three volunteers inhaled each compound.

Evaluation of several solid phase extraction liquid chromatography/tandem mass spectrometry on-line configurations for high-throughput analysis of acidic and basic drugs in rat plasma.

Several configurations using 6- and 10-port switching valves were studied for high flow, on-line extraction of rat plasma coupled to an electrospray triple quadrupole mass spectrometer. Each plasma sample was diluted 1:1 with an aqueous internal standard solution. The sample was injected into a 2.1 x 20 mm cartridge column packed with 25 microm divinylbenzene/N-vinylpyrrolidone packing using 100% aqueous mobile phase at 4 mL/min. After sample loading and sample cleanup, the analytes were eluted from the extraction column with a 1.0-min gradient at 0.4 mL/min. The samples were either analyzed directly after elution from the extraction column or after additional separation using a short high performance liquid chromatography (HPLC) column. The different configurations were tested using an acidic drug (diflunisal) and a basic drug (clemastine) in rat plasma. On-line analysis was performed by injecting 200 microL of diluted plasma. The mass spectrometer was operated in the multiple reaction monitoring (MRM) mode. All calibration standards gave relative standard deviations (RSDs) below 5%. The total time per sample was 3 min.

Simultaneous determination of loxoprofen and its diastereomeric alcohol metabolites in human plasma and urine by a simple HPLC-UV detection method.

A simple, reliable HPLC-UV detection method was developed for the simultaneous determination of loxoprofen and its metabolites (i.e. trans- and cis-alcohol metabolites), in human plasma and urine samples. The method involves the addition of a ketoprofen (internal standard) solution in methanol, zinc sulfate solution and acetonitrile to plasma and urine samples, followed by centrifugation. An aliquot of the supernatant was evaporated to dryness, and the residue reconstituted in a mobile phase (acetonitrile:water=35:65 v/v, pH 3.0). An aliquot of the solution was then directly injected into the HPLC system. Separations were performed on octadecylsilica column (250x4.5 mm, 5 microm) with a guard column (3.2x1.5 cm, 7 microm) at ambient temperature. Loxoprofen and the metabolites in the eluent were monitored at 220 nm (a.u.f.s. 0.005). Coefficients of variations (CV%) and recoveries for loxoprofen and its metabolites were below 10 and over 96%, respectively, in the 200 approximately 15000 ng ml(-1) range for plasma and 500 approximately 50000 ng ml(-1) range for urine. Calibration curves for all the compounds in the plasma and urine were linear over the above-mentioned concentration ranges with a common correlation coefficient of 0.999. The detection limit of the present method was 100 ng for all the compounds. These results indicate that the present method is very simple and readily applicable to routine bioavailability studies of these compounds with an acceptable sensitivity.

Therapeutic drug monitoring of haloperidol, perphenazine, and zuclopenthixol in serum by a fully automated sequential solid phase extraction followed by high-performance liquid chromatography.

In Denmark, haloperidol, perphenazine, and zuclopenthixol are among the most frequently requested antipsychotics for therapeutic drug monitoring. With the number of requests made at the authors' laboratory, the only rational analysis is one that can measure all three drugs simultaneously. The authors therefore decided to develop an automated high-performance liquid chromatography (HPLC) method. Two milliliters serum, 2.0 mL 10 mmol/L sodium phosphate buffer (pH 5.5), and 150 microL internal standard (trifluoperazine) solution were pipetted into HPLC vials and extracted on an ASPEC XL equipped with 1 mL (50 mg) Isolute C2 (EC) extraction columns and acetonitrile-methanol-ammonium acetate buffer (60:34:6) as extracting solution. Three hundred fifty microliters was analyzed by HPLC; a 150 x 4.6-mm S5CN Spherisorb column with a mobile phase of 10 mmol/L ammonium acetate buffer-methanol (1:9), a flow rate of 0.6-1.7 mL/min, and ultraviolet detection at 256 and 245 nm were used. Reproducibility was 5-12% and the lower limit of quantitation was 10, 1, and 5 nmol/L (4, 0.4, and 2 ng/mL) for haloperidol, perphenazine, and zuclopenthixol, respectively. The method was found to be sufficiently selective and robust for routine analysis.

Isotopic Analysis of 28Si-Enriched Silicon Using Laser Mass Spectrometry

A procedure for the isotopic analysis of 28Si-enriched silicon by laser mass spectrometry is described. The procedure is based on the analysis of a dry concentrate or a film obtained by the pneumatic nebulization of an analyte and an internal standard solution or by the laser sputtering of a solid phase onto a support of a high-purity substance. The range of determinable isotope concentrations is (100–n× 10–6) at %; the total error in determining the major isotope is n× 10–2at % at an enrichment factor higher than 99%. The procedure provides the simultaneous control of 70 impurity elements in the sample.

Direct-injection LC–MS–MS method for high-throughput simultaneous quantitation of simvastatin and simvastatin acid in human plasma

A direct-injection liquid chromatography–mass spectrometry–mass spectrometry (LC–MS–MS) method was developed and validated for the simultaneous quantitation in human plasma of the widely used cholesterol-lowering prodrug simvastatin and its in vivo generated active drug, simvastatin acid. The plasma samples were injected into the LC–MS–MS system after simply adding the internal standard solution in an aqueous buffer and centrifuging. The analytes in the buffered plasma samples were found to be stable for at least 24 h at 4°C. The method was successfully validated under the challenging condition of using a large number of quality control (QC) samples including those in which the ratio of the simvastatin concentration to the simvastatin acid concentration was different from the concentration ratio in the calibration curve standards. Under the dual stabilizing conditions of lower temperature (4°C) and lower plasma pH of 4.9, the in-process hydrolysis of simvastatin to simvastatin acid or the lactonization of simvastatin acid to simvastatin was minimized to ≤1.0%. Although the entire run time for on-line cleanup and analysis was only 2.5 min, chromatographic base-line separation of simvastatin from simvastatin acid, which was required to avoid the interference by simvastatin acid with the simvastatin selected reaction monitoring channel, was achieved. The desired lower limit of quantitation of 0.5 ng/ml was achieved by injecting only an equivalent of 8.0 μl of the plasma sample. The extraction column lasted for at least 500 injections.

Nondestructive quantitative determination of docosahexaenoic acid and n−3 fatty acids in fish oils by high‐resolution 1H nuclear magnetic resonance spectroscopy

AbstractBy using a 500 MHz proton nuclear magnetic resonance (1H NMR) spectrometer we have developed a quantitative method for determining the contents of docosahexaenoic acid (DHA) in fish oils (mg/g), the molar proportions (mol%) of DHA to all other fatty acids composing the fish oils, and the molar proportions of total n‐3 fatty acids to all other non‐n‐3 fatty acids in the fish oils. After examining the suitability of ethylene glycol dimethyl ether (EGDM), methanol, and 1,4‐dioxane as internal standards, experimental conditions were optimized by mainly using EGDM as an internal standard. By setting the pulse repetition time at 30 s, five times longer than the longest T1 of the 1H NMR signals of fish oils, good reproductibility of data and analytical times less than 10 min were achieved. The use of the internal standard also allowed us to quantify DHA on a weight basis (mg/g). Verification of the method was carried out in an interlaboratory study between Japan and Norway on bonito, tuna, and salmon oils. The relative errors in the 1H NMR data between Japan and Norway were 0.57–5.29% for quantification of DHA, 0.7–2.09% for the molar proportion of DHA, and 0.1–1.41% for the molar proportion of total n‐3 fatty acids. Good agreement was observed between the 1H NMR data and those obtained by gas chromatography (GC). The sample preparation before 1H NMR measurements required only two steps: sample weighing and preparation of an internal standard solution. Based on the high reproducibility, simplicity of the procedure, and clarity of principle, the proposed 1H NMR method was judged to be a promising alternative to the GC method in quantification of DHA and n‐3 fatty acids in fish oils.

A SIMPLE HPLC METHOD WITH SPECTROPHOTOMETRIC DETECTION FOR THE SIMULTANEOUS ASSAY OF NIFEDIPINE AND VERAPAMIL IN RAT PLASMA

A simple, specific, and accurate HPLC assay method is presented for the simultaneous measurement of the calcium antagonists nifedipine and verapamil in rat plasma. The plasma sample was deproteinized by treatment with an equivalent volume of an internal standard solution (nicardipine hydrochloride in acetonitrile containing 0.1% of perchloric acid), followed by brief centrifugation. A 50 μL aliquot of the clear supernatant was analyzed on a Microsorb-MV C18, 5 μm, column using a mixture of acetonitrile-methanol-0.01 M phosphate buffer pH 5.2 (55:15:30) as the mobile phase. At a flow rate of 1 mL/min and detection at 235 nm, nifedipine, verapamil, and nicardipine were observed to elute at about 3.4 min, 6.4 min, and 9.8 min, respectively. Detector responses were linearly related to concentrations of nifedipine in the range 0.2–1 μg/mL, and of verapamil between 0.4–2 μg/mL. As little as 0.05 μg/mL of nifedipine and 0.08 μg/mL of verapamil were accurately measured by the proposed method. Mean drug recoveries from rat plasma samples spiked with 0.25–1 μg/mL of nifedipine and 0.5–2 μg/mL of verapamil was in all cases >95% (range 97.9–98.3%, n = 5, for nifedipine; 95.7–97.1%, n = 5, for verapamil). The method was found to be well suited for assessing the plasma pharmacokinetics of the title drugs following their oral and intraperitoneal coadministration to rats.