Reference values for serum nitric oxide metabolites in pediatrics

Serum concentration of nitric oxide metabolites (NO_x) is associated with cardiovascular disease risk factors in pediatrics. The aim of this study was to determine sex- and age-specific reference ranges for serum NO_x concentrations in pediatrics. Serum NO_x levels were measured in 401 subjects (189 boys and 212 girls), aged 4–19 years, using the Griess method. Study subjects selected from participants of Tehran lipid and glucose study, an ongoing cohort study aimed at determining of noncommunicable disease risk factors among Tehranian subjects. The International Federation of Clinical Chemistry guidelines and the robust method were used for determining reference values for sample sizes greater or less than 120 respectively. The 95% reference values for serum NO_x concentrations were 13.6–69.2, 11.4–66.0, and 12.2–69.4 μmol/L in boys, girls, and total population respectively. The upper limit of serum NO_x was 28% lower in otherwise healthy overweight and obese boys while it was 6% higher in overweight and obese girls, for both groups compared to their corresponding normal weight subjects. In conclusion, this study, for the first time, reports reference values for serum NO_x levels in healthy children and adolescents.     

Determination of clindamycin in human plasma by liquid chromatography–electrospray tandem mass spectrometry: application to the bioequivalence study of clindamycin

A simple, sensitive and specific liquid chromatography–electrospray tandem mass spectrometry (LC–MS–MS) method for the determination of clindamycin (I) was developed. Both I and verapamil (II, internal standard) were analyzed using a C₁₈ column with a mobile phase of 80% acetonitrile–0.01% trifluoroacetic acid. Column eluents were monitored by electrospray tandem mass spectrometry. Multiple reaction monitoring (MRM) using the parent to daughter combinations of m/z 425→126 and 455→165 was used to quantitate I. A limit of quantitation of 0.0500 μg/ml was found. The assay exhibited a linear dynamic range of 0.0500–20.0 μg/ml and gave a correlation coefficient (r²) of 0.998 or better. The chromatographic run time was approximately 2 min. The intra-batch precision and accuracy of the quality controls (QCs, 0.0500, 0.150, 1.50, 15.0 and 20.0 μg/ml) were characterized by coefficients of variation (CVs) of 5.13 to 13.7% and relative errors (REs) of −4.34 to 4.58%, respectively. The inter-batch precision and accuracy of the QCs were characterized by CVs of 4.35 to 8.32% and REs of −10.8 to −4.17%, respectively. The method has successfully been applied to the analysis of samples taken up to 12 h after oral administration of 300 mg of I in healthy volunteers.     

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.     

Determination of bezafibrate, ciprofibrate and fenofibric acid in human plasma by high-performance liquid chromatography

A selective high-performance liquid chromatographic method to assess either bezafibrate, ciprofibrate or fenofibric acid plasma levels is described. Drugs are extracted with diethyl ether, after acidification with HCl. An isocratic acetonitrile-0.02 M H₃PO₄ (55:45) mobile phase, a C₁₈ (5 μm) column and UV detection are used. The LOQ found was 0.25 μg/ml for the three fibrates. Intra- and inter-assay accuracy ranges were 90–107% and 82–111%; 96–115% and 94–107%; 94–114% and 94–126% for bezafibrate, ciprofibrate and fenofibric acid, respectively. Intra- and inter-assay precision (C.V.% ranges) were 1.72–3.06% and 2.66–7.67%; 1.88–4.64% and 0.62–2.99%; 1.26–4.69% and 3.56–7.17% for the three fibrates studied. Its sensitivity, accuracy and precision make it a useful tool for monitoring plasma levels of these drugs in a clinical setting and for research purposes.     

Urinary 8-hydroxy-2'-deoxyguanosine (8-OHdG) as a marker of oxidative stress in rheumatoid arthritis and aging: effect of progressive resistance training

Urinary 8-hydroxy-2′-deoxyguanosine (8-OHdG), as a measure of oxidative stress, was measured before and after 12 weeks of progressive resistance strength training in 8 healthy elderly (65–80 yr) and eight healthy young (22–30 yr) men and women, and in eight adults (25–65 yr) with rheumatoid arthritis (RA).Training subjects exercised at 80% of their one-repetition maximum and performed eight repetitions per set, three sets per session, on a twice-weekly basis. 8-OHdG was measured at baseline and follow-up (at least 24 hr after the last exercise session) in the RA and elderly subject groups, and at baseline only in young subjects.Baseline 8-OHdG levels were greater among subjects with RA compared to both healthy young (P < 0.001) and elderly (P < 0.05) subjects. There were no changes in 8-OHdG levels in either RA or elderly subjects as a result of the strength training intervention.These results suggest that subjects with RA have higher levels of oxidative stress than young and elderly healthy individuals. Furthermore, there is no change in oxidative stress, measured by urinary 8-OHdG, in elderly healthy individuals or in subjects with RA after a 12-week strength training intervention.     

Polymorphisms of the apolipoprotein B and E genes and their relationship to plasma lipid variables in healthy Chinese men

In this study we have analysed the apolipoprotein (Apo) E polymorphism and polymorphisms of the ApoB gene, including the ApoB/Xba I and ApoB/4311 diallelic polymorphisms and a hypervariable region (HVR) situated in the 3 region of the gene (ApoB/3HVR), in a sample of healthy male subjects from Taiyuan (northern People's Republic of China). In comparison to Caucasian populations, in the Chinese sample, the Xba I2 allele (presence of cutting site; frequency 6.1%; and 95% confidence interval, 3.3–8.9) and the long HVR alleles (9.4%; 6.0–12.8) were rare, whereas the ApoB/4311 (Ser) allele (70.8%; 65.4–76.2) and the 34-repeat allele of the HVR (HVR34; 62.4%; 56.8–68.0) were frequent. In subjects having none, one, or two HVR34 alleles, the mean levels of plasma triglycerides were 2.32±1.44 (SD), 1.45+0.74, and 1.75±1.07 g/l, respectively (P < 0.007). Similar trends were observed for very low density lipoprotein (VLDL) cholesterol, LpE:B, and LpCIII:B. The frequencies of the ApoE alleles were similar to those reported in other populations of Asian origin; E2 (7.4%; 4.2–10.6), E3 (84.4%; 80.2–88.6), and E4 (8.2%; 5.0–11.4). Individuals carrying the E2 allele had a lower mean level of ApoB than E33 individuals: 0.87±0.16 and 1.00±0.22 g/l, respectively (P < 0.007). Individuals carrying the E4 allele had higher levels of ApoE than E33 individuals: 0.140±0.084 and 0.094±0.052 g/l, respectively (P < 0.004); similar trends were observed for VLDL cholesterol, triglycerides, LpE:B, and LpCIII:B. The ApoB/ HVR34 and ApoE/E4 polymorphisms accounted for 10% to 15% of the variability of the plasma levels of VLDL cholesterol, ApoE, triglycerides, LpE:B, and LpCIII:B. Several lipid variables appeared to be favourably affected by specific forms of ApoB and ApoE that are particularly frequent in this Chinese population.     

Enantioselective high-performance liquid chromatographic determination of nicardipine in human plasma

A sensitive method for the enantioselective high-performance liquid chromatography (HPLC) determination of nicardipine in human plasma is described. (+)-Nicardipine, (−)-nicardipine and (+)-barnidipine as an internal standard are detected by an ultraviolet detector at 254 nm. Racemic nicardipine in human plasma was extracted by a rapid and simple procedure based on C₁₈ bonded-phase extraction. The extraction samples were purified and concentrated on a pre-column using a C₁ stationary phase and the enantiomers of nicardipine are quantitatively separated by HPLC on a Sumichiral OA-4500 column, containing a chemically modified Pirkle-type stationary phase. Determination of (+)- and (−)-nicardipine was possible in a concentration range of 5–100 ng ml⁻¹ and the limit of detection in plasma was 2.5 ng ml⁻¹. The recoveries of (+)- and (−)-nicardipine added to plasma were 91.4–98.4% and 93.3–96.7%, respectively, with coefficients of variation of less than 9.0 and 9.4% respectively. The method was applied to low level monitoring of (+)- and (−)-nicardipine in plasma from healthy volunteers.     

Ethyl acetate extraction procedure and isocratic high-performance liquid chromatographic assay for testosterone metabolites in cell microsomes

An isocratic reversed-phase high-performance liquid chromatography (HPLC) method was developed and validated for separation of testosterone and its main metabolites over the nominal range 20 to 40 μg/ml and 280 to 4600 ng/ml, respectively. Mobile phase composition (phosphate buffer–methanol–acetonitrile, 50:38.5:11.5) was optimised by studying the influence of numerous chromatographic parameters. The most critical one was the ratio CH₃CN/CH₃OH. Good recoveries (around 90% for all compounds) and an improved specificity were assessed by a double ethyl acetate extraction of biological samples. According to the performance criteria tested, the method could be applied to enzymatic inhibition and induction in vitro studies.     

Low Serum Testosterone Levels Are Associated with Elevated Urinary Mandelic Acid,and Strontium Levels in Adult Men According to the US 2011–2012 National Health and Nutrition Examination Survey

BackgroundLittle is known regarding the effects of environmental exposure of chemicals on androgenic system in the general population. We studied 5,107 subjects included in the National Health and Nutrition Examination Survey (2011–2012).MethodsUrinary, serum, and blood levels of 15 subclasses comprising 110 individual chemicals were analyzed for their association with serum testosterone levels. The subjects were divided into high and low testosterone groups according to the median testosterone concentration (374.51 ng/dL). Odds ratios (ORs) of individual chemicals in association with testosterone were estimated using logistic regression after adjusting for age, ethnicity, cotinine, body mass index, creatinine, alcohol, and the poverty income ratio.ResultsAdjusted ORs for the highest versus lowest quartiles of exposure were 2.12 (95% CI: 1.07, 4.21; P_trend = 0.044), 1.84 (95% CI: 1.02, 3.34; P_trend = 0.018) for the association between urinary mandelic acid, and strontium quartiles with low testosterone concentrations in adult men, respectively. However, no association was observed for the remaining chemicals with testosterone.ConclusionsThe National Health and Nutrition Examination Survey data suggest that elevations in urinary mandelic acid, and strontium levels are negatively related to low serum testosterone levels in adult men.     

High-performance liquid chromatographic determination of oxolinic acid and flumequine in the live fish feed Artemia

A high-performance liquid chromatography (HPLC) analytical method for the determination of oxolinic acid and flumequine in Artemia nauplii is described. The samples were extracted and cleaned up by a solid-phase extraction (SPE) procedure using SPE C₁₈ cartridges. Oxolinic acid and flumequine were determined by reversed-phase HPLC using a mobile phase of methanol–0.1 M phosphate buffer, pH 3 (45:55, v/v) and a UV detection wavelength of 254 nm. Calibration curves were linear for oxolinic acid in the range of 0.2–50 μg/g (r²=0.9998) and for flumequine in the range of 0.3–50 μg/g (r²=0.9994). Mean recoveries amounted to 100.8% and 98.4% for oxolinic acid and flumequine, respectively. The quantification limit was 0.2 μg/g for oxolinic acid and 0.3 μg/g for flumequine. Quantitative data from an in vivo feeding study indicated excellent uptake of both drugs by Artemia nauplii.     

Control system for detection of the illegal use of naturally occurring steroids in calves

Within the scope of the National Plan for Hormone Control in The Netherlands, a study was performed to develop a system for control of the illegal use of three naturally occurring hormones [oestradiol-17β (E₂-17β), testosterone (T), progesterone (P)] for fattening purposes in animal production. Using a specific high-performance liquid chromatographic—radioimmunoassay method, reference values were established for concentrations of E₂-17β, T and P and some of their metabolites in blood plasma and urine from untreated male and female veal calves. E₂-17β levels of both male and female calves were <0.01 μg/l in blood plasma and <0.2 μg/l in urine. For male veal calves levels of T and epitestosterone (epiT) in blood plasma and urine varied widely. The P levels were <0.1–0.3 μg/l in blood plasma and <0.6–10 μg/l in urine from both male and female calves. To investigate the effect of anabolic treatment on the hormone levels in plasma and excreta, male veal calves were injected, subcutaneously into the dewlap, with a solution containing 20 mg of E₂-17β benzoate and 200 mg of T propionate in 5 ml of arachis oil. Only the levels of E₂-17β and E₂-17α in blood plasma and excreta were elevated until about one week after injection, compared with the untreated control calves and the reference values. T and epiT levels were similar in plasma and excreta from both untreated and treated animals.     

Levels of glutamate,aspartate, GABA,and taurine in different regions of the cerebellum after X-irradiation-induced neuronal loss

The levels of glutamate (Glu), aspartate (Asp), -amino-n-butyric acid (GABA), and taurine (Tau) were determined in the cortex, molecular layer, and deep nuclei of cerebella of adult rats exposed to X-irradiation at 12–15 days following birth (to prevent the acquisition of late-forming granule cells; 12–15x group) and 8–15 days following birth (to prevent the acquisition of granule and stellate cells; 8–15x group). Also, the levels of the four amino acids were measured in the crude synaptosomal fraction (P₂) isolated from the whole cerebella of the control, 12–15x, and 8–15x groups. The level of Glu was significantly decreased by (1) 6–20% in the cerebellar cortex; (2) 15–20% in the molecular layer; and (3) 25–50% in the P₂ fraction of the X-irradiated groups relative to control values. The content of Glu in the deep nuclei was not changed by X-irradiation treatment. Regional levels of Asp were unchanged by X-irradiation, while its level in P₂ decreased by 15–30% after treatment. The levels of GABA and Tau in the molecular layer, deep nuclei, or P₂ were not changed in the experimental groups. However, there was a 15% increase in the levels of GABA and Tau in the cerebellar cortex of the 8–15x group relative to control values. The data support the proposed role of glutamate as the excitatory transmitter released from the cerebellar granule cells but are inconclusive regarding a transmitter role for either Tau or GABA from cerebellar stellate cells.     

Sulfalene concentrations in plasma and blood cells of Plasmodium falciparum malaria cases after treatment with metakelfin using high-performance liquid chromatography

A reversed-phase high-performance liquid chromatographic method using acetonitrile–methanol–1 M perchloric acid–water (25:9:0.8:95, v/v/v) at a flow-rate of 1.0 ml min⁻¹ on LiChrospher 100 RP 18 column (250×4 mm; 5 μm) with UV (254 nm) detection has been developed for the determination of sulfalene in plasma and blood cells after oral administration of the antimalarial drug metakelfin. Calibration curves were linear in the range 0.5–100 μg ml⁻¹. The limit of quantification was 50 ng ml⁻¹. Within-day and day-to-day coefficients of variation averaged 3.84 and 5.31%, respectively. Mean extraction recoveries of sulfalene from plasma and blood cells were 87.21 and 84.65%, respectively. Mean concentrations of sulfalene in plasma of P. falciparum cases on days 2, 7 and 15 were 44.58, 14.90 and 1.70 μg ml⁻¹, respectively; in blood cells concentrations of sulfalene were 7.77, 3.25 and 0.75 μg ml⁻¹, respectively, after oral treatment with two tablets (1000 mg) of metakelfin. Significant difference was recorded on day 2 for sulfalene concentration in blood cells of healthy and P. falciparum cases (t=9.49; P<0.001).     

Analysis of a Panel of 48 Cytokines in BAL Fluids Specifically Identifies IL-8 Levels as the Only Cytokine that Distinguishes Controlled Asthma from Uncontrolled Asthma,and Correlates Inversely with FEV1

We sought to identify cells and cytokines in bronchoalveolar lavage (BAL) fluids that distinguish asthma from healthy control subjects and those that distinguish controlled asthma from uncontrolled asthma. Following informed consent, 36 human subjects were recruited for this study. These included 11 healthy control subjects, 15 subjects with controlled asthma with FEV₁≥80% predicted and 10 subjects with uncontrolled asthma with FEV₁ <80% predicted. BAL fluid was obtained from all subjects. The numbers of different cell types and the levels of 48 cytokines were measured in these fluids. Compared to healthy control subjects, patients with asthma had significantly more percentages of eosinophils and neutrophils, IL-1RA, IL-1α, IL-1β, IL-2Rα, IL-5, IL-6, IL-7, IL-8, G-CSF, GROα (CXCL1), MIP-1β (CCL4), MIG (CXCL9), RANTES (CCL5) and TRAIL in their BAL fluids. The only inflammatory markers that distinguished controlled asthma from uncontrolled asthma were neutrophil percentage and IL-8 levels, and both were inversely correlated with FEV₁. We examined whether grouping asthma subjects on the basis of BAL eosinophil % or neutrophil % could identify specific cytokine profiles. The only differences between neutrophil-normal asthma (neutrophil≤2.4%) and neutrophil-high asthma (neutrophils%>2.4%) were a higher BAL fluid IL-8 levels, and a lower FEV₁ in the latter group. By contrast, compared to eosinophil-normal asthma (eosinophils≤0.3%), eosinophil-high asthma (eosinophils>0.3%) had higher levels of IL-5, IL-13, IL-16, and PDGF-bb, but same neutrophil percentage, IL-8, and FEV₁. Our results identify neutrophils and IL-8 are the only inflammatory components in BAL fluids that distinguish controlled asthma from uncontrolled asthma, and both correlate inversely with FEV₁.     

Determination of 5-hydroxy-N-methylpyrrolidone and 2-hydroxy-N-methylsuccinimide in human urine

A method for simultaneous determination of 5-hydroxy-N-methylpyrrolidone and 2-hydroxy-N-methylsuccinimide in urine is described. These compounds are metabolites of N-methyl-2-pyrrolidone, a powerful and widely used organic solvent. 5-Hydroxy-N-methylpyrrolidone and 2-hydroxy-N-methylsuccinimide were purified from urine by adsorption to a C₈ solid-phase extraction column and then elution by ethyl acetate–methanol (80:20). After evaporation, the samples were derivatised at 100°C for 1 h by bis(trimethylsilyl)trifluoroacetamide. Ethyl acetate was then added and the samples were analysed by gas chromatography–mass spectrometry in the electron impact mode. The extraction recovery for 5-hydroxy-N-methylpyrrolidone was about 80% while that for 2-hydroxy-N-methylsuccinimide was about 30%. The intra-day precision for 5-hydroxy-N-methylpyrrolidone was 2–4% and the between-day precision 4–21% (4 and 60 μg/ml). The intra-day precision for 2-hydroxy-N-methylsuccinimide was 4–8% and the between-day precision 6–7% (2 and 20 μg/ml). The detection limit was 0.2 μg/ml urine for both compounds. The method is applicable for analysis of urine samples from workers exposed to N-methyl-2-pyrrolidone.     

Simultaneous determination of artemether and its major metabolite dihydroartemisinin in plasma by gas chromatography–mass spectrometry-selected ion monitoring

A sensitive, selective, and reproducible GC–MS–SIM method was developed for determination of artemether (ARM) and dihydroartemisinin (DHA) in plasma using artemisinin (ART) as internal standard. Solid phase extraction was performed using C₁₈ Bond Elut cartridges. The analysis was carried out using a HP-5MS 5% phenylmethylsiloxane capillary column. The recoveries of ARM, DHA and ART were 94.9±1.6%, 92.2±4.1% and 81.3±1.2%, respectively. The limit of quantification in plasma was 5 ng/ml (C.V.≤17.4% for ARM and 15.2% for DHA). Calibration curves were linear with R²≥0.988. Within day coefficients of variation were 3–10.4% for ARM and 7.7–14.5% for DHA. Between day coefficients of variations were 6.5–15.4% and 7.6–14.1% for ARM and DHA. The method is currently being used for pharmacokinetic studies. Preliminary data on pharmacokinetics showed C_max of 245.2 and 35.6 ng/ml reached at 2 and 3 h and AUC_0–8h of 2463.6 and 111.8 ngh/ml for ARM and DHA, respectively.     

High-performance liquid chromatographic assay of bromocriptine in plasma and eye tissue of the rabbit

A reliable reversed-phase high-performance liquid chromatographic method has been developed for the determination of bromocriptine (BCT) in plasma and eye tissues. The BCT and propranolol, added as an internal standard (I.S.), were extracted by a liquid–liquid technique followed by an aqueous back-extraction, allowing injection of an aqueous solvent into a 4-μm Nova-Pak C₁₈ column (150×3.9 mm I.D.). The mobile phase was a mixture of 30 parts of acetonitrile and 70 parts of 0.2% triethylamine (pH 3) at a flow-rate of 1 ml/min. Fluorescence detection was at an excitation wavelength of 330 nm and an emission wavelength of 405 nm. The retention times of I.S. and BCT were 4.1 and 11.6 min, respectively. The calibration curve was linear over the concentration range 0.2–10 μg/l for plasma (r>0.999) and vitreous humour (r>0.997) and 1–50 μg/l for aqueous humour (r>0.985). The limit of quantification was 0.2 μg/l for plasma and vitreous humour using a 1-ml sample and was 1 μg/l for aqueous humour using a 0.2-ml sample. The quality control samples were reproducible with acceptable accuracy and precision. The within-day recovery (n=3) was 100–102% for plasma, 91–106% for aqueous humour and 96–111% for vitreous humour. The between-day recovery (n=9) was 90–114% for plasma, 83–115% for aqueous humour and 90–105% for vitreous humour. The within-day precision (n=3) and the between-day precision (n=9) were 1.7–7.0% and 8.1–13.6%, respectively. No interferences from endogenous substances were observed. Taken together, the above simple, sensitive and reproducible high-performance liquid chromatography assay method was suitable for the determination of BCT in plasma and eye tissues following ocular application of BCT for the therapy of myopia.     

Effects of aflatoxin B1 on in vitro cultures ofNicotiana tabacum var. Samsun

Calli ofNicotiana tabacum (tobacco) were treated with two dose ranges of aflatoxin B₁ (0.1–2.0 µg ml^–1 - low does; 5–25 µg ml^–1 aflatoxin B₁). The ability of calli to recover following 3 weeks of toxin exposure was also investigated. The I₅₀ (50% inhibition) value for fresh mass accumulation was approximately 2 µg ml^–1 AFB₁. Fresh mass accumulation was significantly lower than the control value from 0.5 µg ml^–1 AFB₁. Following 3 weeks growth without a toxin source, the growth of calli up to and including 10 µg ml^–1 AFB₁, was significantly greater than control calli, indicating reversibility of the toxic effects. With increasing toxin concentration, chlorophyll content of callus was inhibited from 0.5 µg ml^–1. Transfer to a toxin-free medium resulted in a degree of recovery (up to 0.5 µg ml^–1). In the dose range 5–25 µg ml^–1, the levels of chlorophyll were drastically reduced, with no recovery following AFB₁ removal. Electron microscopy revealed a disruption of chloroplast structure as an early deteriorative event in AFB₁ exposure of callus cells. Protein levels were less sensitive, with inhibition manifested only in the high dose range. Shoot development occurred at all concentrations, but was significantly inhibited from 5 µg ml^–1 AFB₁. Recovery following toxin removal was minimal at these higher AFB₁ concentrations. The number of necrotic calli increased progressively from 5 µg ml^–1 as toxin levels increased.     

Quantitative determination of cefepime in plasma and vitreous fluid by high-performance liquid chromatography

An isocratic reversed-phase HPLC method was developed to determine cefepime levels in plasma and vitreous fluid. Cefepime and the internal standard cefadroxil were separated on a Shandon Hypersil BDS C18 column by using a mobile phase of 25 mM sodium dihydrogen phosphate monohydrate (pH 3) and methanol (87:13, v/v). Ultraviolet detection was carried out at 270 nm. The retention times were 4.80 min for cefepime and 7.70 min for cefadroxil. This fast procedure which involves an efficient protein precipitation step (addition of HClO₄), allows a quantification limit of 2.52 μg ml⁻¹ and a detection limit of 0.83 μg ml⁻¹. Recoveries and absolute recoveries of cefepime from plasma were 96.13–99.44% and 94–102.5% respectively. The intra-day and inter-day reproducibilities were less than 2% for cefepime at 10, 30, 50 μg ml⁻¹ (n=10).The method was proved to be suitable for determining cefepime levels in human plasma and was modified to measure vitreous fluid samples.     

Rapid and sensitive gas-chromatographic determination of caffeine in blood plasma,saliva, and xanthine beverages

A gas chromatographic procedure is reported for the determination of caffeine in plasma, saliva, and xanthine beverages. Using a 75 cm column packed with OV-17, nitrogen-sensitive detection, and 1 ml samples, a suitable limit of analysis (coefficient of variation (CV)=10.2%) of 50 ng/ml was obtained in plasma. Within-day CVs at caffeine concentrations of 0.1–0.5–2.0–7.5–15.0 g/ml in plasma were 7.7–5.6–4.8–3.8–3.4%, respectively. The limit of detection, defined as the injected quantity of caffeine giving rise to a signal to noise ratio of 2, is 40 pg, corresponding to a plasma concentration of 1 ng/ml.The procedure involves addition of the internal standard 7-pentyl theophylline and alkaline extraction of the sample with dichloromethane. The method described rivals any gaschromatographic assay published so far in rapidness and accuracy.Plasma and saliva caffeine concentrations were determined in a healthy male volunteer after swallowing 400 ml of coffee. The calculated pharmacokinetic parameters, assuming complete absorption of caffeine from the G.I. tract, agree well with previously published values.