An improved high-performance liquid chromatography method for the determination of homocysteine in human plasma

Summary Elevated plasma homocysteine is, a known risk factor in arteriosclerotic vascular disease. To measure homocysteine in a large number of samples, we have developed a rapid, simple, robust and inexpensive reversed-phase HPLC method for routine analysis. Mercaptopro-pionylglycine was used as the internal standard and an external calibration in plasma was performed. Improvement was achieved by the use of gradient elution (using a sodium acetate buffer and methanol) resulting in a higher number of samples analyzed per day. Plasma samples were reduced with tributylphosphine and the proteins were precipitated with perchloric acid before addition of internal standard. The analytes were derivatized by use of 7-fluorobenzofurazone-4-sulfonic acid ammonium salt. For calibration human plasma was spiked with nine different concentrations of homocysteine (range 2–50 μmol L⁻¹). The inter-assay precision of replicate (n=29) analysis of the concentration of homocysteine in a sample of pooled plasma was 3.0%. The limit of detection, defined as three times the signal-to-noise ratio, was 0.25 μmol L⁻¹. The linearity of the assay was confirmed for a plasma concentration range of 2–2000 μmol L⁻¹. The variation of duplicate analyses of 842 plasma samples was 2.6±1.7%.     

An improved analytical method for determination of heterocyclic amines in chicken legs

Summary Several extraction, separation and detection methods for heterocyclic amines (HAs) in chicken legs were evaluated by liquid chromatography. Results showed that the most appropriate extraction method includes the removal of macrosubstances by centrifugation and subsequent purification using a PRS (propylsulfonic acid silica gel) and a C₁₈ cartridge, and the recovery obtained ranged between 51 and 89 %. For HPLC separation, a binary solvent system consisting of acetonitrile and 0.05 M ammonium acetate solution (pH 3.6) with gradient elution with flow rate of 1.0 mL min⁻¹ and detection at 258 nm was used to resolve 16 HAs. With fluorescence nine HAs could be detected by employing a programmable wavelength, and the sensitivity was 100–400 times higher than that by UV detection. The detection limits for UV and fluorescence detection were 0.02≈0.5 ng and 0.05≈3 pg respectively, with a signal-to-noise ratio 3. The presence of HAs in fried chicken legs was also determined.     

An improved method for the determination of free sphingosine in serum

Summary A rapid, sensitive and selective high-performance liquid chromatographic method has been developed for the determination of sphingosine in human serum. After precipitation with methanol, the samples were extracted using Carbopack B disposable columns; the sphingosine was eluted with 0.05 M hydrochloric acid in methanol-dichloromethane (20∶80, v/v) and the extract evaporated to dryness at 40°C. The sample residue was then reconstituted with methanol and reacted with o-phthaldialdehyde reagent to produce a fluorescent compound. Separation was performed using an LC-18 column with 0.05 M phosphate buffer (pH 7)-methanol-acetonitrile (15∶80∶5, v/v) as mobile phase. Fluorescence detection was performed with excitation and emission wavelengths of 340 and 455 nm, respectively. The serum extract was re-analyzed with a cyano LC column to minimize the possibility of false positive results. The possible interference of compounds having a structure similar to that of sphingosine was evaluated. The mean recovery of sphingosine was >94.5%. The limit of detection of the assay was 1 ng mL⁻¹. The between-run and within-run coefficients of variation for replicate analyses were <4.0% and <3.4%, respectively. The levels of free sphingosine in the serum of 40 normal subjects (20 male and 20 female) was investigated; the average level was 81.6±41.1 ng mL⁻¹ (mean ±S.D.) for males and 85.5±33.7 ng mL⁻¹ for females.     

On line trace enrichment for isolation of vitamin B2 in food samples

Summary Vitamin B₂ was enriched by liquid-solid extraction from large volumes of aqueous samples on a short precolumn. The enriched compounds were transferred onto an analytical reversed-phase column and separated by ion-pair chromatography. The equipment used provides the possibility of automation for routine analysis.Dedicated to Professor J. F. K. Huber on the occasion of the 60th birthday.     

Development and validation of an improved liquid chromatographic method for the analysis of oxytetracycline

Summary An improved LC method is described for the separation of oxytetracycline and its impurities. The separation is much better than that obtained with official pharmacopoeia methods. The method uses XTerra RP-18, 5 μm (25cm×4.6 mm I.D.), a silica-based stationary phase with methyl end-capping, claimed to reduce silanol activity. The column temperature is set at 30°C and a UV detection is performed at 280 nm. Mobile phase containing acetonitrile −0.25 M tetrabutylammonium hydrogen sulfate pH 7.5−0.25 M ethylenediaminetetraacetic acid pH 7.5-water (115:360:160:365,v/v/v/v) is used at a flow rate of 1.0 mL.min⁻¹, to separate the impurities present in oxytetracycline base. A central composite experimental design is used to optimize the separation. A second isocratic method with higher content of acetonitrile is needed to separate the more retained impurities present only in oxytetracycline hydrochloride. The method is robust and shows good selectivity, repeatability, linearity and sensitivity.     

A validated HPLC method for assay of morphine hydrochloride and hydromorphone hydrochloride in pharmaceutical injections

Summary A sensitive and rapid routine HPLC method is proposed for quantitative estimation of morphine hydrochloride and hydromorphone hydrochloride in pharmaceutical dosage forms. The drugs were chromatographed on a C₁₈ reversed-phase column; the mobile phase was acetonitrile-water, 35:65 (v/v), containing sodium dodecyl sulphate (0.5%, w/v), as ion pairing reagent, and acetic acid (0.4% v/v). Detection was at 230 nm. The optimized method was validated and linearity (r>0.999), precision, and accuracy were found to be acceptable within the concentration ranges 86–124 μg mL⁻¹ for morphine hydroloride and 60–180 μg mL⁻¹ for hydromorphone hydrochloride. The method is being used to investigate the stability of morphine hydrochloride and hydromorphone hydrochloride in solution used for intramuscular injection.     

An improved HPLC-ED method for monitoring plasma levels of clozapine and its active metabolites in schizophrenic patients

Summary An HPLC method with electrochemical detection has been developed for the determination of clozapine and its main metabolites, desmethylclozapine and clozapine N-oxide, in human plasma. An accurate pretreatment of the biological samples was implemented by means of solid phase extraction (SPE) on HLB cartridges. This improved pretreatment, together with a new mobile phase, allows for the accurate determination of clozapine N-oxide, which could not be quantitated by a previous method. The method uses only 100 μL of plasma for one complete analysis and shows good recovery values for all three analytes. The eluates from the SPE procedure were chromatographed in a reversed phase C18 column using a mobile phase composed of phosphate buffer, acetonitrile and methanol. Clozapine, desmethylclozapine and clozapine N-oxide were eluted in less than 10 minutes, without any interference from the biological matrix. Linearity was observed over the 2.50–150 ng mL⁻¹ (clozapine and desmethylclozapine) or 1.25–75 ng mL⁻¹ clozapine N-oxide) range for the three analytes, with satisfactory repeatability values. The limit of detection was 0.3 ng mL⁻¹ for clozapine and desmethylclozapine, samples of patients treated with Leponex gave good results. No interference from other common central nervous system drugs was found. This method seems to be a useful tool for pharmacokinetic studies and for clinical monitoring, because of its need for small plasma samples and its high sensitivity and selectivity.     

An improved method for the determination of S-Carboxymethyl-L-cysteine in biological fluids with precolumn derivatization and on-line clean-up

Summary In order to follow levels of S-Carboxymethyl-L-cysteine in biological fluids for a period as long as three half-lives after drug administration during pharmacokinetic studies, an improved method for its determination had to be developed. Like the previous one, this method uses a protein precipitation step followed by an O-Phthalaldehyde derivatization step and then an HPLC on-line clean-up. This latter was obtained by means of a switching valve system, including a Nucleosil CN 5 m (3 cm × 4.6 mm i.d.) precolumn and a Spherisorb ODS 5 m (15 cm×4.6 mm i.d.) analytical column. The sensitivity limit was improved to 0.1 g/ml in plasma samples and 0.2 g/ml in urine samples.This method was applied in studies comparing single (0.75 g) and repeated (0.75 g tid) oral administration of the drug to 30 elderly patients and 20 healthy volunteers. Results showed that the half-life was 40% longer in elderly patients than in healthy volunteers, and that area under the plasma concentration versus time curve (AUC) values in elderly patients were twice those obtained with young subjects.     

A validated, stability-indicating method for the assay of dexamethasone in drug substance and drug product analyses, and the assay of preservatives in drug product

Summary A new high-performance liquid chromatographic (HPLC) procedure for the determination of dexamethasone, imprities, degradation products and product preservatives is described. A three-stage, linear gradient with UV detection at 240 nm allows the alysis of dexamethasone drug substance and dexamethasone in two formulated products, using the same chromatographic system. The Limit of Quantitation (LOQ) of dexamethasone importies in drug substance is 0.05%, and 0.1% for dexamethasone degradation products in formulated products. The method is linear, precise, accurate and robust. Sample preparations are simiple, and are accomplished without the use of an internal standard. Several degradation products of stressed dexamethasone have been identified.     

An improved HPLC system for the analysis of photosynthetic pigments

Summary A high performance liquid chromatography system for the analysis of photosynthetic pigments is presented. The method employs an octadecylsilica stationary phase, a programmed quaternary mobile phase consisting of mixtures of methanol, acetonitrile, water and hexane, and a photodiode array detector. Carotenoids and chlorophylls are rapidly analysed in a single chromatographic separation. Thecis-trans isomers of most carotenoids are separated by this method.     

Method for determination of plasticizers in industrial emissions

Summary A simple method has been developed for the determination of dibutyl phthalate, di-(2-ethylhexyl) phthalate, diisodecyl phthalate, and di(2-ethylhexyl) adipate in industrial emissions. A cartridge packed with a modified silica gel is used as adsorbent and adsorbed compounds are eluted and analyzed by HPLC in normal and reversed phase system.Presented at the 2nd Conference on Solid Phase Extraction, Bratislava, Soovakian Republic, November 16–18, 1992.     

Chromatographic and spectral behaviour and detection of hepatotoxic nodularin in fish, clam, mussel and mouse tissues using HPLC analysis

Summary High-performance liquid chromatography (HPLC) was applied in the analysis of nodularin (NODLN), a potent, bioaccumulable hepatotoxin. The behaviour of NODLN in biological matrices and possibility to analyse biota samples for NODLN content was examined using a conventional HPLC/diode array detector method that uses C₁₈ solid-phase cartridge clean up. Tissues of European flounder, blue mussel (spiked and naturally contaminated), clam (exposed to NODLN in an aquariuml and mouse (subjected to i. p. administration of NODLN) were analysed. UV detection was 5 times more sensitive than electrochemical detection. Recovery of NODLN from spiked tissues was 59% for mussel, 53% for flounder, and 44–75% for mouse tissues. NODLN was detected in clams exposed with NODLN, but not in naturally contaminated mussels where NODLN conjugation occurs. Through the use of spectral processing, free NODLN was unambiguously identified from tissue samples. The HPLC method showed limits of quantification between 90 and 150 μg NODLN kg⁻¹ dw. The method proved applicable for routine tissue analysis and can be used in the monitoring of acutely toxic NODLN levels.     

An enhanced liquid chromatographic method for 5-hydroxymethylfurfural determination in UHT milk

Summary An enhanced method of HPLC for the determination of 5-Hydroxymethyl-2-furfuraldehyde (HMF) in fluid milk was perfected. Its resolving capacity permitted the specific separation of this compound. The real concentration (mol/l) of HMF was determined and not the HMF value, as in the colorimetric analysis, where an intermediary coloured compound (HMF-TBA) is utilised. The optimum pH condition (pH=4.0–4.2) for the mobile phase to prevent overlapping between the HMF peak and the peaks of the other compound ( and ) that coeluted with the HMF and also the optimum organic solvent content (3% or 7%) were established. This is a sensitive, quick and safe method for the determination of HMF in fluid milk.Research funded by CICYT n. ALI-173.     

A stability-indicating high-performance liquid chromatographic assay for the determination of some pharmaceutically important nitrocompounds

Summary A simple, rapid and precise stability-indicating high performance liquid chromatographic assay for the determination of furazolidone (I), nitrofurazone (II), nitrofurantoin (III), niridazole (IV) and nifuroxime (V) in pure form and in pharmaceutical preparations has been devloped.Reversed phase chromatography was conducted using a Lichrosorb R.P. 18 column (250×4 mm), with methanol-water-buffer pH3 (40555) eluent and detection at 365, 375, 367, 368 and 340 nm respectively. Calibration graphs were linear over the concentration ranges 3–18, 1–10, 1–10, 3–18 and 3–18 m·ml^–1. Reoveries from bulk drugs were 99.94–100.00 with a relative standard deviation of 1.02–1.61.Sunlight degradation of most of the studied drugs to 5-nitrofuraldehyde was observed. Accelerated stability studies have been carried out on each drug by exposure to sunlight for different time periods. Graphs of log of the remaining concentration (Log. C) against time indicated that photodecomposition of the nitrocompounds is a first order reaction. The proposed method was applied to some representative dosage forms and the results obtained were in good agreement with those obtained by the official methods.     

RP-HPLC assay for 1,2-5,6-dianhydro-3,4-disuccinyl-galactitol and its metabolites in blood plasma and liver

Summary A method involving pre-column derivatization and HPLC assay is described for measuring submicrogram quantities of 1,2-5,6-dianhydro-3,4-disuccinyl-galactitol (1,2-5,6-dianhydro-3,4-bis(carboxypropionyl)-galactitol), an effective cytostatic drug and its metabolites in blood plasma and liver homogenate. The substance and its metabolites were derivatized with sodium pentamethylene-dithiocarbamate to form different bis(dithiocarbamoyl) esters, which can be detected by UV absorbance at 254 and 280 nm. The directly derivatized products were then extracted into CHCl₃, and after sample preparation resolved by RP-HPLC on SAS-Hypersil column.     

Highly sensitive method for the determination of a new anthracycline: Pirarubicin

Summary A new and highly sensitive HPLC method for the simultaneous determination of pirarubicine (THP-doxorubicin) and its metabolites, adriamycin and adriamycinol, in human plasma, is described. Samples were treated by liquid-liquid extraction, the organic phase removed and the residue dissolved in methanol. Separation was on a Lichrocart Supersher RP 8 column, (250×4 mm) 4 m, with a mobile phase of acetonitrile/methanol/formate-buffer.     

An improved high-performance liquid chromatographic determination of chlorpropamide in human plasma

Summary Samples were extracted with dichloromethane and the organic layer evaporated to dryness. The residue was dissolved in methanol, and 10 μl aliquot injected onto the column. Tolbutamide was used as the internal standard for chlorpropamide. The UV detector response was linear over the range 0–200 μg ml⁻¹ with a correlation coefficient of 0.999; detection limit: 0.002 μg ml⁻¹. Within-day and between-day assay variation was generally ≤7%. No interference from endogenous constituents was observed. The utility of the method was demonstrated by determining chlorpropamide in samples from six healthy volunteers following a single oral dose of 250 mg. The procedure is simple and requires small volumes of plasma.     

An optimized direct method for the determination of carboxylic acids in beverages by HPLC

Summary A direct method for the simultaneous determination of tartaric, malic, lactic, acetic, citric, shikimic, fumaric and succinic acids in fruit juices and wines by isocratic reversed phase HPLC is reported.The variables (pH, ionic strength, flow and temperature) have been optimized by a modification of the original simplex method. The separation factor (s) and calibrated resolution product (r^*) have been used as criteria for selectivity optimization. After validation, the method has been applied to the determination of carboxylic acids in apple, orange and lemon juices, white and red wines and musts during the fermenation process.     

Analysis of vitamin E in food and phytopharmaceutical preparations by HPLC and HPLC-APCI-MS-MS

Summary The analysis of α, β, γ, δ-tocopherols, trienols, α-tocopheryl acetate and nicotinate (vitamin E) in complex matrices was carried out using a new liquid chromatographic (HPLC) method giving better separation efficiency, selectivity and sensitivity than that described in the literature. The use of normal-phase (NP)-HPLC on silica gel with issoctane-diisopropylether-1,4-dioxane as optimized mobilepphase yielded higher resolution than conventional reversed-phase (RP)-HPLC using methanol mobile phase. Identification of peaks was by UV-absorbance at 295 nm, diode array, or fluorescence detection (λ _ex = 295 nm,λ _ex = 330 nm). The latter was found to be more selective and ten times more sensitive than UV-absorbance detection. A quadrupole, ion-trap mass spectrometer with an atmospheric-pressure ionization (APCl) interface was used to detect vitamin E constituents in the femtomole range. With collision-induced dissociation (CID) in the ion source, which gave characteristic fragmentation, the identity of the investigated compounds could be confirmed. Plots of peak area versus amount injected allowed quantitation of α, β, γ, δ-tocopherols and-trienols, α-tocopheryl acetate and nicotinate in real samples such as peanut, almond, spinach, spelt grain bran, latex and tablets. The method described offers fast identification and quantitation of vitamin E constituents of complex biological origin. Dedicated to Professor Dr. Heinz Engelhardt on the occasion of his 65^th birthday.