Simple and rapid method for the measurement of nitrite and nitrate in human plasma and cerebrospinal fluid by capillary electrophoresis

Nitrite and nitrate levels in physiological fluids are commonly used as an index of nitric oxide production. We developed simple and rapid method for the determination of these anions by capillary zone electrophoresis employing borate buffer (pH 10, 100 mmol/l) as running electrolyte. The anions were analyzed in plasma and cerebrospinal fluid (CSF) without deproteinization of the samples. Electrophoresis was carried out in a capillary (36.5 cm×75 μm) at a potential of 15 kV, with on-column UV detection at 214 nm. Mean retention times for nitrite and nitrates were 4.631 and 5.152 min, respectively. The method was linear (r=0.999) within a 1–500 μmol/l concentration range. Physiological levels of nitrate in plasma (40.2 μmol/l) and CSF (15.3 μmol/l) could be determined with good precision (coefficients of variation <6%) and accuracy (recoveries of added nitrate to plasma and CSF were 97.4 and 104.5%, respectively). Measurements of the physiological levels of nitrite in plasma (6.1 μmol/l) and CSF (0.9 μmol/l) were less precise and accurate.     

Determination of monobromobimane derivatives of phenylmercapturic and benzylmercapturic acids in urine by high-performance liquid chromatography and fluorimetry

A method was developed for the determination in human urine of S-phenylmercapturic (PMA) and S-benzylmercapturic (BMA) acids, metabolites respectively of benzene and toluene. PMA and BMA were determined, after alkaline hydrolysis, to give respectively thiophenol and benzylmercaptan, and coupling of the thiol-containing compounds with monobromobimane (MB), by reversed-phase HPLC on a diphenyl-silica bonded cartridge (100×4.6 mm I.D., 5 μm particle size) with fluorimetric detection. Wavelengths for excitation and emission were 375 and 480 nm, respectively. The recovery of PMA and BMA from spiked urines was >90% in the 10–500 μg/l range; the quantification limits were respectively 1 and 0.5 μg/l; day-to-day precision at 42 μg/l was C.V. <7%. The suitability of the proposed procedure for the biological monitoring of exposure to low-level airborne concentrations of benzene and toluene, was evaluated by analyzing the urinary excretion of PMA and BMA in subjects exposed to different sources of aromatic hydrocarbons, namely occupationally-unexposed referents (non-smokers, n=15; moderate smokers, n=8; mean number of cigarettes smoked PER-DAY=17 cig/day) and non-smoker workers occupationally exposed to toluene in maintenance operations of rotogravure machines (non-smokers, n=17). Among referents, non-smokers showed values of PMA ranging from <1 to 4.6 μg/l and BMA from 1.0 to 10.4 μg/l; in smokers, PMA values ranging from 1.2 to 6.7 μg/l and BMA from 9.3 to 39.9 μg/l, were observed. In occupationally exposed non-smoker subjects, BMA median excretion value (23.6 μg/l) was higher than in non-smoker referents (3.5 μg/l) (P<0.001) and individual BMA values (y, μg/l) were associated and increased with airborne toluene concentration (x, mg/m³) according to the equation y=6.5+0.65x (r=0.69, P<0.01, n=17). The proposed analytical method appears to be a sensitive and specific tool for biological monitoring of low-level exposure to benzene and toluene mixtures in occupational and environmental toxicology laboratory.     

A method for low volume and low Se concentration samples and application to paired cerebrospinal fluid and serum samples

The well-known beneficial health effects of Se have demanded the development of rapid and accurate methods for its analysis. A flow injection (FI) method with inductively coupled plasma mass spectrometry (ICP-MS) as a selenium-selective detector was optimized. Flow injection was carried out using a Knauer 1100 smartline inert series liquid chromatograph coupled with a Perkin Elmer DRC II ICP-mass spectrometer. For sample injection a Perkin Elmer electronic valve equipped with a 25 μL sample loop was employed. Before measurement, standards or samples were administered with 1 μg/L rhodium as internal standard for correction of changes in detector response according to changes in sample electrolyte concentration. The method characterization parameters are: LOD (3σ criterion): 26 ng/L, LOQ (10σ criterion): 86 ng/L, linearity: 0.05–>10 μg/L, r²=0.9999, serial or day-to-day precision at 2 μg/L: 4.48% or 5.6%. Accuracy was determined by (a) recovery experiments (CSF spiked with 2 μg/L Se); (b) comparison of FI-ICP-MS measurement with graphite furnace atomic absorption (GFAAS) measurements of 1:10 diluted serum samples; (c) Se determination in urine and serum control materials. Recovery (a) was 101.4%, measurement comparison with GFAAS (b) showed 98.8% (5 serum samples, 1:10 diluted in the range of 0.5–1.3 μg/L, compared to GFAAS determination, which was set to 100%), and accuracy was 96.8% or 105.6% for the serum or urine control material. Analysis time per sample was short and typically below 2 min for the complete measurement, including sample introduction, sample-line purge and quadruplicate Se determination.This method was used to determine Se in cerebrospinal fluid (CSF) and plasma (here parallel to GFAAS) in 35 paired serum and CSF samples. Se determination gave values in the range of 42–130 μg/L for serum and 1.63–6.66 μg/L for CSF. The median for Se in 35 individual CSF samples was 3.28 μg/L, the mean (±SD) was 3.67 (1.35) μg/L, whilst for individual serum samples the median was 81 μg/L and the mean (±SD) was 85 (26) μg/L. When relating the paired Se concentrations of CSF samples to respective serum samples it turned out that Se-CSF (behind blood brain barrier (BBB)) is independent on Se-serum concentration (before BBB).     

Sensitive and specific high-performance liquid chromatographic assay with ultraviolet detection for the determination of cocaine and its metabolites in rat plasma

A sensitive, specific and precise HPLC–UV assay was developed to quantitate cocaine (COC) and its metabolites benzoylecgonine (BE), norcocaine (NC) and cocaethylene (CE) in rat plasma. After adding 50 μl of the internal standard solution (bupivacaine, 8 μg/ml) and 500 μl of Sørensen's buffer (pH 6) to 100 μl of rat plasma sample, the mixture was extracted with 10 ml of chloroform. The organic layer was transferred to a clean test tube and was evaporated under nitrogen. The residue was reconstituted in 100 μl of mobile phase and 35 μl was injected onto the HPLC column. The mobile phase consisted of methanol–acetonitrile–50 mM monobasic ammonium phosphate (5:7:63, v/v/v) and was maintained at a flow-rate of 0.4 ml/min. Separation of COC and its metabolites was achieved using a Supelcosil ABZ+plus deactivated reversed-phase column (250×2.1 mm I.D., 5 μm). Calibration curves were linear over the range of 25–5000 ng/ml for COC and its three metabolites. The absolute extraction efficiencies for BE, COC, NC, CE and bupivacaine were 56.6%, 78.6%, 61.1%, 76.4% and 67.0%, respectively. COC and its metabolites were stable in mobile phase for 24 h at room temperature and in rat plasma for 2 weeks at −20°C. The limits of detection for BE, COC, NC and CE were 20, 24, 15 and 12.9 ng/ml, respectively. These values correspond to 0.70, 0.84, 0.525 and 0.452 ng of the according compound being injected on column. The within-day coefficient of variation for the four compounds ranged from 3.0% to 9.9% while the between-day precision varied from 3.6% to 14%. This method was used to analyze rat plasma samples after administration of COC alone and in combination with alcohol. The pharmacokinetic profiles of COC and its metabolites in these rats are also described.     

Determination of mycophenolic acid and its phenol glucuronide metabolite in human plasma and urine by high-performance liquid chromatography

Simultaneous determination of mycophenolic acid (MPA) and mycophenolate phenol glucuronide (MPAG) in plasma and urine was accomplished by isocratic HPLC with UV detection. Plasma was simply deproteinated with acetonitrile and concentrated, whereas urine was diluted prior to analysis. Linearity was observed from 0.2 to 50 μg/ml for both MPA and MPAG in plasma and from 1 to 50 μg/ml of MPA and 5 to 2000 μg/ml MPAG in urine with extraction recovery from plasma greater than 70%. Detection limits using 0.25 ml plasma were 0.080 and 0.20 μg/ml for MPA and MPAG, respectively. The method is more rapid and simple than previous assays for MPA and MPAG in biological fluids from patients.     

Ion interaction reagent reversed-phase high-performance liquid chromatography determination of anti-tuberculosis drugs and metabolites in biological fluids

New methods of ion interaction reagent (IIR) RP-HPLC are presented for the determination of anti-tuberculosis drugs and their metabolites, singly or in multi-component mixtures, in biological fluids. The following analytes are considered: isoniazid, ethionamide, pyrazinamide, morphazinamide, p-aminosalicylic acid, nicotinic and isonicotinic acids. Aqueous solutions of three different ion interaction reagents are alternatively or comparatively used as the mobile phases, namely: (A) 5.00 mM octylamine at pH 3.00 for o-phosphoric acid, (B) 5.00 mM octylamine at pH 8.00 for o-phosphoric acid, and (C) 5.00 mM 1,6 diaminohexane at pH 6.00 for o-phosphoric acid. The response linearity between peak area and analyte concentration is verified for all the analytes in the concentration range within the determination limits and 2.00 mg/l. Detection limits are always lower than 82 μg/l for standard solutions; in the analysis of samples of rat serum, rat plasma and human serum, the matrix effect is negligible, the detection limits are always lower than 94 μg/l and the average recovery yield is always greater than 96%.     

Use of novel solid-phase extraction sorbent materials for high-performance liquid chromatography quantitation of caffeine metabolism products methylxanthines and methyluric acids in samples of biological origin

An automated reversed-phase high-performance liquid chromatographic (RP-HPLC) method, using a linear gradient elution, is described for the simultaneous analysis of caffeine and metabolites according to their elution order: 7-methyluric acid, 1-methyluric acid, 7-methylxanthine, 3-methylxanthine, 1-methylxanthine, 1,3-dimethyluric acid, theobromine, 1,7-dimethyluric acid, paraxanthine and theophylline. The analytical column, an MZ Kromasil C₄, 250×4 mm, 5 μm, was operated at ambient temperature with back pressure values of 80–110 kg/cm². The mobile phase consisted of an acetate buffer (pH 3.5)–methanol (97:3, v/v) changing to 80:20 v/v in 20 min time, delivered at a flow-rate of 1 ml/min. Paracetamol was used as internal standard at a concentration of 6.18 ng/μl. Detection was performed with a variable wavelength UV–visible detector at 275 nm, resulting in detection limits of 0.3 ng per 10-μl injection, while linearity held up to 8 ng/μl for most of analytes, except for paraxanthine and theophylline, for which it was 12 ng/μl and for caffeine for which it was 20 ng/μl. The statistical evaluation of the method was examined performing intra-day (n=6) and inter-day calibration (n=7) and was found to be satisfactory, with high accuracy and precision results. High extraction recoveries from biological matrices: blood serum and urine ranging from 84.6 to 103.0%, were achieved using Nexus SPE cartridges with hydrophilic and lipophilic properties and methanol–acetate buffer (pH 3.5) (50:50, v/v) as eluent, requiring small volumes, 40 μl of blood serum and 100 μl of urine.     

Determination of BAY 12-8039, a new 8-methoxyquinolone, in human body fluids by high-performance liquid chromatography with fluorescence detection using on-column focusing

A reversed-phase (RP) high-performance liquid chromatographic (HPLC) method with fluorescence detection allowing the sensitive and specific quantification of BAY 12-8039, a new antimicrobially active 8-methoxyquinolone, in biological fluids is described. The method is compared to a microbiological assay (bioassay) based on B. subtilis test strain with a limit of quantification of approximately 60 μg/l. Following dilution and centrifugation, plasma, saliva or urine supernatant is directly injected onto the HPLC system. Concentrations down to a limit of quantification of 2.5 μg/l can be quantified in plasma, saliva and urine. Data on recovery, accuracy and precision of the method throughout the whole working range as well as results on stability of the analyte are presented. The concentration data are correlated with results from the bioassay. BAY 12-8039 is stable in plasma after repeated freeze-thaw cycles and following storage at −20°C for at least 12 months. The results of HPLC measurements excellently agree with bioassay data indicating the relevance of the method as a tool in clinical development to answer pharmacokinetic questions related to antimicrobial activity. The method was applied to human plasma, saliva and urine from subjects after a single oral dose of 400 mg of BAY 12-8039.     

Capillary electrophoresis analysis of nitrite and nitrate in sub-microliter quantities of airway surface liquid

We developed a simple capillary electrophoresis (CE) method to measure nitrite and nitrate concentrations in sub-microliter samples of rat airway surface liquid (ASL), a thin (10–30 μm) layer of liquid covering the epithelial cells lining the airways of the lung. The composition of ASL has been poorly defined, in large part because of the small sample volume (1–3 μl per cm² of epithelium) and difficulty of harvesting ASL. We have used capillary tubes for ASL sample collection, with microanalysis by CE using a 50 mM phosphate buffer (pH 3), with 0.5 mM spermine as a dynamic flow modifier, and direct UV detection at 214 nm. The limit of detections (LODs), under conditions used, for ASL analysis were 10 μM for nitrate and 30 μM for nitrite (S/N=3). Nitrate and nitrite were also measured in rat plasma. The concentration of nitrate was 102±12 μM in rat ASL and 70±1.0 μM in rat plasma, whereas nitrite was 83±28 μM in rat ASL and below the LOD in rat plasma. After instilling lipopolysaccharide intratracheally to induce increased NO production, the nitrate concentration in ASL increased to 387±16 μM, and to 377±88 μM in plasma. The concentration of nitrite increased to 103±7.0 μM for ASL and 138±17 μM for plasma.     

The role of prostaglandins in the excitatory innervation of the rat urinary bladder

The possible role of PGs in hyoscine-resistant nerve mediated responses of the rat urinary bladder was investigated. Responses to electrical stimulation were inhibited by cinchocaine (30 μmol/l) but were only partially inhibited by a high concentration of hyoscine (25 μmol/l) or by the choline uptake inhibitors, hemicholinium-3 (500 μmol/l) and troxypyrrolidinium (500 μmol/l). Indomethacin (50 μmol/l) produced partial blockade (30%) of responses to electrical stimulation without markedly affecting responses to acetylcholine and the degree of blockade was of a similar order in the presence of hyoscine or troxypyrrolidinium. PGE₂ (0.028 – 2.8 μmol/l) or F_2α (0.029 – 2.9 μmol/l) produced a slowly developing increase in tone and spontaneous activity. Responses to electrical stimulation were at most only slightly increased in the presence of either PG. However, the PGs always increased the responses to electrical stimulation after indomethacin, indomethacin plus hyoscine or indomethacin plus troxypyrrolidinium. Responses to acetylcholine in the presence of indomethacin were not increased by PGE₂. It is concluded that PGE₂ and F_2α do not function as transmitters responsible for resistance to anti-muscarinic drugs in the bladder but may exert a modulating effect on nervous transmission.     

Determination of plasma phenobarbital concentration by high-performance liquid chromatography in rat offspring

Plasma phenobarbital (PB) concentrations in rat offspring were determined using a 9 μl capillary by high-performance liquid chromatography (HPLC). Capillary plasma which was put into a Bond Elut^® cartridge column by using 1 ml of 0.01 M KH₂PO₄ was applied to the column with 50 μl of 2 μg/ml of acetanilide (internal standard, I.S.). After washing the column, PB and I.S. were eluted with methanol and injected into the HPLC system. There were excellent linear correlation between the amount of PB and length of the capillary at three different concentrations. Calibration for PB was linear in the range of 0–50 μg/ml. The coefficients of variation were 3.4–5.0% and 5.9–7.5% in the within-day and between-day assays, respectively. The extraction recovery rates were 87.5–105.4%. By this method, it was possible to measure plasma PB concentrations in rat offspring without killing. These results suggested that this method is very useful to determine the plasma PB concentration derived from mother’s milk in newborn rats.     

Determination of pyrazinamide and its main metabolites in rat urine by high-performance liquid chromatography

A new high-performance liquid chromatograhic procedure for simultaneous determination of pyrazinamide (PZA) and its three metabolites 5-hydroxypyrazinamide (5-OH-PZA), pyrazinoic acid (PA), and 5-hydroxypyrazinoic acid (5-OH-PA), in rat urine was developed. 5-OH-PZA and 5-OH-PA standards were obtained by enzymatic synthesis (xanthine oxidase) and checked by HPLC and GC–MS. Chromatographic separation was achieved in 0.01 M KH₂PO₄ (pH 5.2), circulating at 0.9 ml/min, on a C₁₈ silica column, at 22°C. The limits of detection were 300 μg/l for PZA, 125 μg/l for PA, 90 μg/l for 5-OH-PZA and 70 μg/l for 5-OH-PA. Good linearity (r²>0.99) was observed within the calibration ranges studied: 0.375–7.50 mg/l for PZA, 0.416–3.33 mg/l for PA, 0.830–6.64 mg/l for 5-OH-PZA and 2.83–22.6 mg/l for 5-OHPA. Accuracy was always lower than ±10.8%. Precision was in the range 0.33–5.7%. The method will constitute a useful tool for studies on the influence of drug interactions in tuberculosis treatment.     

Determination of flumequine and 7-hydroxyflumequine in plasma of sheep by high-performance liquid chromatography

A high-performance liquid chromatographic (HPLC) method for the simultaneous determination of flumequine and its metabolite 7-hydroxyflumequine in sheep plasma was described. The two compounds were extracted from 100 μl of plasma by liquid–liquid extraction. Aliquots (100 μl) were injected onto the HPLC system and separated on a LiChrospher Select B column with an isocratic system. The compounds were detected by fluorimetric detection for concentrations below 500 μg/l and by UV detection for the concentrations exceeding 500 μg/l. The range of the validated concentrations were 50 000 to 5 μg/l and 500 to 10 μg/l with mean recovery rates of 87±3% and 60±1% for flumequine and 7-hydroxyflumequine, respectively.     

Analysis of pipecolic acid in biological fluids using capillary gas chromatography with electron-capture detection and [H11]pipecolic acid as internal standard

A sensitive and accurate stable isotope dilution assay was developed for the measurement of pipecolic acid in body fluids using capillary gas chromatography with electron-capture detection. The method utilizes [²H₁₁]pipecolic acid as the internal standard. Sample preparation consisted of derivatization in aqueous solution (pH 11.5) of the amine moiety with methyl chloroformate to the N-methylcarbamate, followed by acidic ethyl acetate extraction at pH ≤ 2 and further derivatization of the carboxyl moiety with pentafluorobenzyl bromide, the excess of which was removed by solid-phase extraction. Control values have been determined in the plasma of at-term infants, age > 1 week (n = 21, mean = 1.36 μM, range = 0.47–3.27 μM). The utility of the method was demonstrated by quantitating pipecolic acid in biological fluids derived from patients with peroxisomal disorders. The method was validated against an established electron-capture negative ion mass fragmentographic technique.     

Measurement and pharmacokinetic study of tetramethylpyrazine in rat blood and its regional brain tissue by high-performance liquid chromatography

We used a rapid, sensitive and reliable high-performance liquid chromatographic method for the determination of tetramethylpyrazine in rat brain tissue and plasma. The lower limit of quantification in plasma and brain tissue was 0.1 μg/ml and 0.1 μg/g, respectively, and only a small amount of plasma (100 μl) or brain tissue (100 μg) was required for analysis. The decline in the concentration of tetramethylpyrazine in plasma was generally two-exponential at a dose of 2, 5, or 10 mg/kg administered intravenously. Concentrations of tetramethylpyrazine in various regions of the brain (cerebral cortex, brainstem, striatum, hippocampus, cerebellum and midbrain) were not significantly different at 15 min following drug administration (10 mg/kg, i.v.). In additional analysis, mean concentration of the tetramethylpyrazine in rat plasma was approximately five-times greater than the drug in brain tissue.     

Dual microcolumn immunoassay applied to determination of insulin secretion from single islets of Langerhans and insulin in serum

A dual microcolumn immunoassay (DMIA) was developed and applied to determination of insulin in biological samples. The DMIA utilized a protein G capillary column (150 μm I.D.) with covalently attached anti-insulin to selectively capture and concentrate insulins in a sample. Insulins retained in the capillary immunoaffinity column were desorbed and injected onto a reversed-phase capillary column (150 μm I.D.) for further separation from interferences such as cross-reactive antigens and non-specifically adsorbed sample components. Bovine, porcine and rat insulin all cross-reacted with the antibody and could be determined simultaneously. Using a UV absorbance detector, the dual microcolumn system had a detection limit of 10 fmol or 20 pM for 500-μl sample volumes. The DMIA system was used to measure glucose-stimulated insulin secretion from single rat islets of Langerhans. Because of the separation in the second dimension, both rat I and rat II insulin could be independently determined. The system was also evaluated for determination of insulin in serum. Using microcolumns instead of conventional HPLC columns resulted in several advantages including use of less chromatographic material and improved mass detection limit.     

Determination of temozolomide in human plasma and urine by high-performance liquid chromatography after solid-phase extraction

As a part of a pilot clinical study, a high-performance reversed-phase liquid chromatography analysis was developed to quantify temozolomide in plasma and urine of patients undergoing a chemotherapy cycle with temozolomide. All samples were immediately stabilized with 1 M HCl (1 + 10 of biological sample), frozen and stored at −20°C prior to analysis. The clean-up procedure involved a solid-phase extraction (SPE) of clinical sample (100 μl) on a 100-mg C₁₈-endcapped cartridge. Matrix components were eliminated with 750 μl of 0.5% acetic acid (AcOH). Temozolomide was subsequently eluted with 1250 μl of methanol (MeOH). The resulting eluate was evaporated under nitrogen at RT and reconstituted in 200 μl of 0.5% AcOH and subjected to HPLC analysis on an ODS-column (MeOH-0.5% AcOH, 10:90) with UV detection at 330 nm. The calibration curves were linear over the concentration range 0.4–20 μg/ml and 2–150 μg/ml for plasma and urine, respectively. THe extraction recovery of temozolomide was 86–90% from plasma and 103–105% from urine over the range of concentrations considered. The stability of temozolomide was studied in vitro in buffered solutions at RT, and in plasma and urine at 37°C. An acidic pH (<5–6) shoul be maintained throughout the collection, the processing and the analysis of the sample to preserve the integrity of the drug. The method reported here was validated for use in a clinical study of temozolomide for the treatment of metastatic melanoma and high grade glioma.     

Cell-free DNA in Human Follicular Microenvironment: New Prognostic Biomarker to Predict in vitro Fertilization Outcomes

Cell-free DNA (cfDNA) fragments, detected in blood and in other biological fluids, are released from apoptotic and/or necrotic cells. CfDNA is currently used as biomarker for the detection of many diseases such as some cancers and gynecological and obstetrics disorders. In this study, we investigated if cfDNA levels in follicular fluid (FF) samples from in vitro fertilization (IVF) patients, could be related to their ovarian reserve status, controlled ovarian stimulation (COS) protocols and IVF outcomes. Therefore, 117 FF samples were collected from women (n = 117) undergoing IVF/Intra-cytoplasmic sperm injection (ICSI) procedure and cfDNA concentration was quantified by ALU-quantitative PCR. We found that cfDNA level was significantly higher in FF samples from patients with ovarian reserve disorders (low functional ovarian reserve or polycystic ovary syndrome) than from patients with normal ovarian reserve (2.7 ± 2.7 ng/μl versus 1.7 ± 2.3 ng/μl, respectively, p = 0.03). Likewise, FF cfDNA levels were significant more elevated in women who received long ovarian stimulation (> 10 days) or high total dose of gonadotropins (≥ 3000 IU/l) than in women who received short stimulation duration (7–10 days) or total dose of gonadotropins < 3000 IU/l (2.4 ± 2.8 ng/μl versus 1.5 ± 1.9 ng/μl, p = 0.008; 2.2 ± 2.3 ng/μl versus 1.5 ± 2.1 ng/μl, p = 0.01, respectively). Finally, FF cfDNA level was an independent and significant predictive factor for pregnancy outcome (adjusted odds ratio = 0.69 [0.5; 0.96], p = 0.03). In multivariate analysis, the Receiving Operator Curve (ROC) analysis showed that the performance of FF cfDNA in predicting clinical pregnancy reached 0.73 [0.66–0.87] with 88% specificity and 60% sensitivity. CfDNA might constitute a promising biomarker of follicular micro-environment quality which could be used to predict IVF prognosis and to enhance female infertility management.     

Simultaneous determination of six penicillins in cows'' raw milk by a multiresidue high-performance liquid chromatographic method

A high-performance liquid chromatographic method based on C₁₈ solid-phase extraction and ultraviolet detection at 323 nm of analytes derivatized with benzoic anhydride and 1,2,4-triazole mercuric chloride solution was developed for the simultaneous determination of amoxicillin, penicillin G (benzylpenicillin), ampicillin, oxacillin, cloxacillin and dicloxacillin residues in raw milk. The detection limit of the method was 1.3 μg/l for penicillin G; 1.4 μg/l for amoxicillin, oxacillin and cloxacillin, 1.5 μg/l for ampicillin and 2.7 μg/l for dicloxacillin. The mean recovery was 95–102% for amoxicillin, penicillin G and ampicillin, 92–98% for oxacillin and cloxacillin and 87–94% for dicloxacillin, measured by using an internal standard. The relative repeatability standard deviation was 4–9% on level 4–15 μg/l, respectively, 2–7% on level 30–40 μg/l.     

Rapid determination of ethylene glycol at toxic levels in serum and urine

The rapid gas chromatographic detection and determination of ethylene glycol in biological fluids is described. Phenylboronic acid in acetone was used for the esterification of glycol. The phenylboronates of ethylene glycol and 1,2-propylene glycol are not separated on a packed column of medium polarity (OV-17), but they can be separated on a non-polar column (OV-101). In both instances 1,3-propylene glycol can be used as an internal standard. The method requires only 100 μl of serum or urine and is suitable for trace analysis in an emergency toxicological laboratory. The utility of the method is demonstrated on two cases of human intoxication with ethylene glycol.