Candidate reference method: Determination of valproic acid in serum by isotope-dilution mass spectrometry

Summary A method has been developed for the determination of valproic acid, without derivatization, in human serum by isotope-dilution mass spectrometry using labelled 2-propyl[3,3,3-d₃]valeric[5,5,5-d₃] acid as internal standard for accurate quantification of the concentration of valproic acid in the sample. After acidification, the analyte and internal standard are extracted withn-hexane. The amounts of valproic acid in the serum are calculated from the isotope ratio of valproic acid to labelled valproic acid, which is measured by electron impact (EI) and chemical ionization (CI) selected ion monitoring (SIM). The concentrations of valproic acid in sera measured using isotope-dilution mass spectrometry are compared with results from gas-liquid chromatography (GLC) and fluorescence polarization immunoassay (FPIA). The accuracy, precision and recovery of the GC-MS methods are discussed. The coefficient of variation determined from duplicate samples was less than 1.5%. The detection limit was 10 ng mL⁻¹ at a signal-to-noise ratio of 3∶1. Part of this work was presented at the Kongre? der Deutschen Gesellschaft für Laboratoriumsmedizin, Berlin, 1994.     

Candidate reference method: Determination of valproic acid in serum by isotope-dilution mass spectrometry

Summary A method has been developed for the determination of valproic acid, without derivatization, in human serum by isotope-dilution mass spectrometry using labelled 2-propyl[3,3,3-d₃] valeric[5,5,5-d₃] acid as internal standard for accurate quantification of the concentration of valproic acid in the sample. After acidification, the analyte and internal standard are extracted withn-hexane. The amounts of valproic acid in the serum are calculated from the isotope ratio of valproic acid to labelled valproic acid, which is measured by electron impact (EI) and chemical ionization (CI) selected ion monitoring (SIM). The concentrations of valproic acid in sera measured using isotope-dilution mass spectrometry are compared with results from gas-liquid chromatography (GLC) and fluorescence polarization immunoassay (FPIA). The accuracy, precision and recovery of the GC-MS methods are discussed. The coefficient of variation determined from duplicate samples was less than 1.5%. The detection limit was 10 ng mL⁻¹ at a signal-to-noise ratio of 3:1. Part of this work was presented at the Kongre? der Deutschen Gesellschaft für Laboratoriumsmedizin, Berlin, 1994.     

HPLC Determination of Valproic Acid in Human Serum Using Ultraviolet Detection

Abstract

A sensitive HPLC method with minimal sample preparation and good reproducibility for the determination of valproic acid in serum is described. Serum samples were precipitated using acetonitrile containing diazepam as the internal standard. Chromatography was performed on a Hewlett Packard model 1090 equipped with an octadecylsilane column and a Beckman model 163 variable wavelength detector. The drug and internal standard were eluted isocratically using a mobile phase consisting of 0.01M sodium phosphate monobasic solution, pH 2.3 and acetonitrile (63:37 v/v) followed by a gradient to flush the column before the next sample injection. The flow rate was 2.5 mL/min, the injection volume was 25 μL and the effluent was monitored at 210 nm. The serum standard curve was linear from 2.5-200.0 μg/mL with a correlation coefficient of 0.9994. Day-to-day precision for quality control samples (10.0, 25.0, 75.0 μg/mL serum) ranged from 5.6-9.6% CV. Possible interferences from other drugs which might be administered concurrently were studied. The method has been applied to the analysis of human serum samples.     

Glc Determination of Valproic Acid in Plasma

Abstract

A one-step glc procedure was developed for the quantitative determination of valproic acid in plasma. After addition of internal standard (4-methyl valeric acid), plasma is buffered at pH 4.5 to avoid hydrolysis of valproic acid conjugate (s). Evaporation is avoided by extraction into a chloroform bead. The method is sufficiently sensitive to quantitate 0.8 μg of the drug in 0.2 ml of plasma. The procedure has been thoroughly tested for precision and reproducibility through the determination of over two thousand samples.     

HPLC determination of valproic acid in human plasma by derivatization with O-p-nitrobenzyl-N,N′-diisopropylisourea

A sensitive and specific method for the determination of valproic acid in plasma has been developed. After the proteins in the plasma have been precipitated with a saturated solution of ammonium sulfate in 1N HCl, the valproic acid, together with the internal standard, is extracted from the plasma with dichloromethane. An aliquot of the organic solution is taken for derivatization of the valproic acid and the internal standard with O-p-nitrobenzyl-N,N′-diisopropylisourea. Separation is carried out by HPLC using two chromatographic systems: an adsorption system with a μ Porasil column, hexane-chloroform (94:6) as mobile phase, and caproic acid as internal standard and a partition reverse phase system comprising a μ Bondapak T_M/C₁₈ column, acetonitrile/methanol/0.0035 M phosphate buffer (60:10:30), and caprylic acid as internal standard. UV detection is at 254 nm. This method, developed in both systems, permits the determination of plasma levels of valproic acid in the reported range of 50-100 μg/mL. With adequate sensitivity, specificity, precision, and accuracy. The plasma levels of valproic acid may be determined by this method without interference from the commonest antiepileptic drugs. Good correlation is obtained with the enzymatic immune analytic method: EMIT.     

Simultaneous Determination of Six Anticonvulsants in Serum by High Performance Liquid Chromatography

Abstract

The authors describe a simultaneous determination method of six anticonvulsants (ethosuximide, primidone, phenobarbital, phenytoin, carbamazepine, valproic acid) using 500 μl of serum.

After the addition of the internal standard (5-(p-methylphenyl)-5 phenylhydantoin), the anticonvulsants are extracted in an acid medium with ethyl acetate. They are separated by reverse phase chromatography on a μ Bondapack C18 column, eluted with a water/methanol mixture (36/64 V/V) at a flow rate of 0.7 ml/min.

The column effluent is first analyzed by monitoring the ultraviolet absorption at 197 nm and then at 425 nm after the addition of a color indicator: bromocresol purple. The analysis lasts 12 minutes at ambient temperature. The sensitivity obtained with the serum for the range of products investigated is of the order of 0.5 to 2 mg/l, the extraction recoveries varying from 75 to 100% depending on the drug. Reproducibility is good (cv ≤ 9%).     

A novel freeze‐dried storage and preparation method for the determination of mycophenolic acid in plasma by high‐performance liquid chromatography

Plasma samples were conventionally stored at freezing conditions until the time of detection. Such a technique, when carried out over an extended period, is energy consuming; in addition, preparation and transportation of stored samples is inconvenient. In this study, a freeze‐dried storage and preparation method was proposed to determine the presence of mycophenolic acid (MPA) in plasma. Fresh plasma samples were freeze‐dried using a device, and then stored at ambient temperature. After the stored samples were soaked with methanol spiked with the internal standard, high‐performance liquid chromatography was conducted to detect MPA. The proposed method was demonstrated to be precise and accurate over the linear range of 0.5–50 μg mL⁻¹, with both intra‐ and inter‐day precision being <7% and biases <10%. The freeze‐dried samples were stable at ambient temperature for at least 40 days. This method was also successfully applied to the pharmacokinetic study of MPA in healthy volunteers. Pharmacokinetic parameters, such as maximum plasma concentration, time point of maximum plasma concentration and elimination half‐life, among others, were consistent with the results in the published study. This proposed technique was proved to be simple, reproducible and energy saving. This approach could also simplify the storage and analysis of samples in clinical and scientific drug research.     

Enzyme Immunoassay of Thyroid-Stimulating Hormone Using Dried Blood Samples a Simple Technique of Screening for Congenital Hypothyroidism

Abstract

A method for enzyme imnunoassay of thyroid-stimulating hormone (TSH) in dried blood spotted onto filter paper has been developed. TSH was conjugated to horse-radish peroxidase according to Nakane's method. Separation of the bound and free fractions was obtained by a double antibody solid phase method using polyacetal beads which were coated with the purified IgG fraction from goat anti-rabbit IgG serum. p-Hydroxyphenyl propionic acid was used as substrate for the fluorophotometric assay of peroxidase activity. The assay sensitivity is 0.07, μU TSH/assay tube, which is equivalent to μU/ml when five 3 mm discs of dried blood spot are assayed. TSH values in dried blood samples obtained by this method correlate well with those of serum samples obtained by radioimmunoassay (r=0.89). The coefficients of variation were 6.8 to 13.4% (within assay) and 5 to 40% (between assay). The enzyme immunoassay of TSH presented here is applicable to the mass-screening for congenital hypothyroidism of neonate.     

Rapid Determination of Methotrexate and 7-Hydroxymethotrexate in Serum and Cerebrospinal Fluid by Radial Compression Liquid Chromatography

Abstract

A rapid, specific and sensitive radial compression reverse phase liquid chromatographic method for the analysis of methotrexate and 7-hydroxymethotrexate in serum and cerebrospinal fluid is reported. A mobile phase consisting of acetonitrile-methanol-pH 3 phosphate (8:15:77) at 6 ml/min flow rate was employed. The U.V. detector was set at 317 nm, and folic acid was used as an internal standard. A rapid extraction of methotrexate and 7-hydroxymethotrexate was performed using Sep-Pak cartridges with high extraction efficiency for both compounds. Patients serum and cerebrospinal fluid samples were analyzed by the described method and the concentrations of methotrexate were compared to those obtained by an enzyme immunoassay. No interference from other metabolites or anticancer drugs in the described assay was observed.     

Determination of Bromine in Human Serum by Inductively Coupled Plasma-Mass Spectrometry

Abstract

The determination of bromine in human serum by inductively coupled plasma-mass spectrometry (ICP-MS) is discussed. Sample preparation was kept as limited as possible: serum samples were only diluted with nitric acid (five- or ten-fold). Indium was added as internal standard. In order to avoid overlap of ⁴⁰Ar ⁴⁰Ar with     

Evaluation and Application of the Internal Standard Technique for the Direct Determination of Copper in Fruit Juices Employing Fast Sequential Flame Atomic Absorption Spectrometry

Abstract

The present paper describes the evaluation and application of internal standard for the determination of copper in fruit juices, employing Fast Sequential Flame Atomic Absorption Spectrometry (FS FAAS). The internal standards tested were indium, cobalt, and nickel using correlation graphs. However, indium was used, considering the composition of the samples. After this step, copper was determined in fruit juices using indium as internal standard. This method allows the determination of copper with a limit of quantification of 0.011 mg L^?1. The fruit juice samples selected for analysis were of grape, orange, pineapple, peach, cashew, and strawberry. The contents of copper in these samples varied from 0.02 to 0.42 mg L^?1. The analytical results were compared with the results obtained by analysis of these samples after complete mineralization using acid digestion and determination employing FS FAAS. The statistical comparison by a t-test (95% confidence level) showed no significant difference between the results. The relative standard deviations (RSD) with and without the use of the internal standard for a copper solution containing 0.4 mg L^?1 were of 0.62 and 1.94%, respectively. The use of indium as internal standard provided more accurate analytical results, as well as better analytical performance for the determination of copper in juice samples.     

Studies on target tissue distribution of ginsenosides and epimedium flavonoids in rats after intravenous administration of Jiweiling freeze‐dried powder

A simple and rapid liquid chromatography–mass spectrometry (LC‐MS) method was developed and validated for analysis of ginsenoside Rb₁, Rb₂, Rc, Rd, Re, Rf, Rg₁, icariin and epimedin A, B, C in rat target tissues (spinal cord, brain, muscle and sciatic nerve) after intravenous administration of Jiweiling freeze‐dried powder using genistein as an internal standard (IS). The tissue samples were treated by protein precipitation with methanol prior to HPLC and chromatographic separation was performed on a C₁₈ column utilizing a gradient elution program with acetonitrile and 0.1% formic acid aqueous. Electrospray ionization (ESI) source was employed and the 11 analytes and IS were detected by multiple reaction monitoring (MRM) scanning under the negative ionization mode. Higher sensitivity was achieved and the optimized mass transition ion‐pairs (m/z) for quantitation were selected. The calibration curves were linear over the investigated concentration ranges with correlation coefficients higher than 0.995. The intra‐ and inter‐day RSDs were all less than 10% with the relative error (RE) within ±9.3%. The mean extraction recoveries for all compounds were between 93.3 and 106%. The proposed method was successfully applied to investigate the target tissue distribution of the 11 compounds in rat after intravenous administration of Jiweiling freeze‐dried powder. Copyright © 2011 John Wiley & Sons, Ltd.     

Liquid chromatography/electrospray ionization mass spectrometry method for the quantification of valproic acid in human plasma

A simple, sensitive and rapid liquid chromatography/electrospray ionization mass spectrometry (LC/ESI-MS) method was developed and validated for the quantification of valproic acid, an antiepileptic drug, in human plasma using benzoic acid as internal standard (IS). Following solid-phase extraction, the analytes were separated using an isocratic mobile phase on a reversed-phase C18 column and analyzed by MS in the single ion monitoring mode using the respective [M-H]- ions, m/z 143 for valproic acid and m/z 121 for the IS. The assay exhibited a linear dynamic range of 0.5-60 microg/mL for valproic acid in human plasma. The lower limit of quantification was 500 ng/mL with a relative standard deviation of less than 10%. Acceptable precision and accuracy were obtained for concentrations over the standard curve range. The average absolute recoveries of valproic acid and the IS from spiked plasma samples were 96.1+/-4.2 and 95.6+/-2.7%, respectively. A run time of 4.5 min for each sample made it possible to analyze more than 250 human plasma samples per day. The validated method has been successfully used to analyze human plasma samples for application in pharmacokinetic, bioavailability and bioequivalence studies.     

BCR (non-certified) reference materials for dioxins and furans in milk powder

Milk powders spiked with different quantities of polychlorinated dibenzo-p-dioxins (PCDD) and dibenzofurans (PCDF) have been prepared by both freeze drying and spray drying. In 1991, the freeze dried batches were used in a BCR intercomparison. The spray dried batches have now been compared to the freeze dried ones. The obtained results show that:

-there is no significant difference between the materials;

-the recoveries of the added amounts of D48, D54, D66, D67, D70, F83, F94, F114, F118, F121 and F130 are of the order of 90%.

On this basis, assigned values are proposed for the spray dried materials which will become available as a set of three (non-certified) BCR reference materials (RM 532, RM 533, RM 534).     

Determination of Organophosphorus Insecticides in Water by C-18 Solid Phase Extraction and Quantitative TLC

Abstract

A simple, rapid, and sensitive method for the determination of sarafloxacin (A-55620) in fish serum using enrofloxacin as internal standard is described. The serum sample and internal standard enrofloxacin are loaded onto the extraction column packed with C2 sorbent material. The column is rinsed and then eluted. The detection limit is 5 ng/g, the recovery rate varying from 92 to 100 % .     

Rapid Determination of Coumatetralyl in Human Serum by High‐Performance Liquid Chromatography Coupled with Electrospray Ionization Tandem Mass Spectrometry

Abstract

A rapid, sensitive, and selective high‐performance liquid chromatography‐tandem mass spectrometric method (HPLC‐MS‐MS) for the determination of coumatetralyl in human serum using warfarin as an internal standard has been developed and validated. Coumatetralyl and the internal standard were extracted from the human serum samples by liquid‐liquid extraction with ethyl acetate, followed by separation on a XDB C₁₈ reversed‐phase column (150 mm×2.1 mm i.d., 5 µm) using a mobile phase consisting of acetic acid‐ammonium acetate (5 mmol/L, pH=4.5)/methanol (20:80, v/v) at a constant flow rate of 0.40 mL/min. Coumatetralyl and the internal standard were ionized by negative ion pneumatically assisted electrospray and detected in the multiple‐reaction monitoring mode using precursor→product ion combinations at m/z 291→247 and 307→161, respectively. The calibration curve was linear (r²=0.9945) in the concentration range of 0.5～100.0 ng/mL, with a lower limit of quantification of 0.5 ng/mL in human serum. Intra‐ and inter‐day relative standard deviations were less than 6.3 and 11.0%, respectively. The mean extraction recovery was 87.9% for coumatetralyl and 90.1% for the internal standard. This method is found to be able to determine trace coumatetralyl in human serum and can be used for the diagnosis of poisoned human beings.     

Simultaneous Determination of Carbamazepine,Phenobarbital and Their Major Metabolites in Serum,Brain Tissue and Urine by High-Performane Liquid Chromatographya

Abstract

A high-performance liquid chromatographic method is described for the simultaneous measurement of carbamazepine, phenobarbital and their major metabolites in small samples of serum, brain tissue and urine. This involves solvent extraction, reversed-phase chromatography and ultraviolet detection at 195 nm. Glucuronides in urine are hydrolyzed by enzymatic cleavage with β-glucuranidase. Quantitation is based on the peak-height ratio of the analyte to its internal standard (10, 11-dihydrocarbamaze-pine or p-methylphenobarbital). The results obtained show that the method is precise and reproducible.     

Quantitation of Serotonin in Human Plasma,Serum and Cerebrospinal Fluid Samples by HPLC-EC Using 6-Hydroxytryptamine as an Internal Standard

Abstract

A simple method for sample clean up and concentration of serotonin (5-HT) in biological samples, such as human cerebro-spinal fluid and serum, is described. To the sample 6-hydroxy-tryptamine (6-HT) is added as an internal standard and it is then absorbed either on C₁₈ SEP-PAK cartridge or Biorex-70 short column. 5-HT and 6-HT are then eluted from the column with methanolic formic acid. After evaporation, the residue is dissolved in the mobile phase and an aliquot is used for LC-EC quantitation.     

Liquid Chromatographic Assay for Amodiaquine in Tablets and Biological Fluids

Abstract

A high performance liquid chromatographic assay for quantitating amodiaquine (A) in tablets, urine, plasma, bile and saliva is described. The method involved acid extraction of the drug from tablets and chloroform extraction of its base from the biological fluids after alkalinization with ammonia. Quinidine was used as the internal standard. A μ-Bondapak phenyl column was used for separation together with a mobile phase made of methanol, water and glacial acetic acid (pH 2.3). Good chromatograms with efficient separation of drug and internal standard peaks were observed. Retention times of 5.2 and 7.1 min. were obtained for the drug and the internal standard respectively. Correlation between areas under the curve and drug concentration was high. The mean percentage recovery of A from tablets was 102.03%, while from the biological fluids, it ranged from 85.2 to 104.61%. Urine and saliva obtained from volunteers and bile obtained from animals administering amodiaquine showed chromatograms similar to those obtained for blank samples spiked with A. Interference from table't excipients of biological fluids was undetectable or negligible. The method was found to be precise and simple.     

Determination of lithium in biological samples by inductively coupled plasma mass spectrometry

Lithium was determined in human serum by inductively coupled plasma mass spectrometry. Sample preparation was kept to the minimum: serum samples were diluted and beryllium was added as internal standard. Special attention was given to the choice of the internal standard and to the occurrence of memory effects. To test the accuracy of the method several biological reference materials were analysed, namely a “Second-Generation” Biological Reference Material (Freeze-Dried Human Serum) (University of Ghent), Human Serum SRM 909, Whole Egg Powder SRM 1845 and Mixed Human Diet SRM 1548 (National Institute of Standards and Technology). The results were compared with those obtained by other techniques. For the “second-generation” reference freeze-dried human serum a mean lithium concentration of 15.10 ng g^?1 with a standard deviation of 0.54 ng g^?1 dry weight was found. Analyses on serum samples from healthy individuals yielded lithium concentrations ranging from 0.22 to 0.97 μg l^?1.