Determination of ethyl glucuronide in human serum by hyphenation of capillary isotachophoresis and zone electrophoresis

The determination of ethyl glucuronide (EtG), a marker of recent alcohol consumption, in human serum by hyphenation of capillary ITP (CITP) and CZE is reported. For CITP step, 1 x 10(-2) M hydrochloric acid adjusted with epsilon-aminocaproic acid (EACA) to pH 4.4 was used as the leading electrolyte, and 1 x 10(-2) M nicotinic acid with EACA, pH 4.4, was used as the terminating electrolyte (TE). All electrolytes contained 0.2% hydroxypropylcellulose to suppress electroosmosis. In CITP, EtG was separated from fast serum macrocomponents chloride, phosphate, lactate, and acetate. Zones of microcomponents including EtG that migrated between acetate and nicotinate were forwarded to the second capillary filled with a BGE identical with the TE. Conductivity detection was used in the CITP step. Sensitive detection in the CZE step was performed using indirect spectrophotometric detection at 254 nm. The assay is based on a 1:5 dilution of serum with deionized water and has a concentration LOD for EtG in diluted sample of 9.8 x 10(-9) M. The method was used for the determination of EtG in sera of volunteers consuming alcohol.     

Analysis of ethyl glucuronide in human serum by capillary electrophoresis with sample self-stacking and indirect detection

Ethyl glucuronide (EtG), a metabolite of ethanol, is a marker of recent alcohol consumption. In the past few years, its analysis in body fluids has attracted considerable attention because it closes a gap between short time and long time alcohol markers such as ethanol and carbohydrate-deficient transferrin, respectively. The capillary zone electrophoresis (CZE) analysis of EtG in model mixtures and human serum is reported using uncoated and coated fused-silica capillaries together with acidic buffers in the pH range between 3.2 and 4.4 and indirect detection. In these approaches, separation of EtG from endogenous macro- and microcomponents (anionic serum components of high and low concentration, respectively) is based upon transient isotachophoretic stacking referred to as sample self-stacking. The selection of a favorable bufferco-ion and pH is shown to be crucial for optimized sensitivity. Abuffercomposed of 10 mM nicotinic acid and epsilon-aminocaproic acid (pH 4.3) is demonstrated to provide a detection limit for EtG in serum of 0.1 microg/ml, a value that is relevant for clinical and forensic purposes.     

Determination of ethyl glucuronide in human serum by capillary zone electrophoresis and an immunoassay

Ethyl glucuronide (EtG) is a marker of recent alcohol consumption. For the optimization of the analysis of EtG by CZE with indirect absorbance detection, the use of capillaries with permanent and dynamic wall coatings, the composition of the BGE, and various sample preparation procedures, including dilution with water, ultrafiltration, protein precipitation, and SPE, were investigated. Two validated screening assays for the determination of EtG in human serum, a CZE‐based approach and an enzyme immunoassay (EIA), are described. The CZE assay uses a coated capillary, 2,4‐dimethylglutaric acid as an internal standard, and a pH 4.65 BGE comprising 9 mM nicotinic acid, ε‐aminocaproic acid and 10% v/v ACN. Proteins are removed via precipitation with ACN prior to analysis and the LOQ is 0.50 mg/L. The EIA is based upon commercial reagents which are promoted for the determination of urinary EtG. Krebs–Ringer solution containing 5% BSA is used as a calibration matrix. All samples are ultrafiltered prior to analysis of the ultrafiltrate on a Mira Plus analyzer. Assay calibration ranged between 0 and 2 mg/L and the upper reference limit was determined to be 0.05 mg/L. Both assays proved to be suitable for the analysis of samples from different individuals. For EtG levels above 0.50 mg/L, good agreement was observed for the comparison of the results of the two methods.     

Confirmation analysis of ethyl glucuronide and ethyl sulfate in human serum and urine by CZE-ESI-MS(n) after intake of alcoholic beverages

CZE coupled to sheath liquid-based electrospray ionization (ESI) and multiple-stage ion trap mass spectrometry (MS(n) ) was used for the confirmation analysis of ethyl glucuronide (EtG) and ethyl sulfate (EtS) in human serum and urine collected after intake of alcoholic beverages. Electrophoretic separations were performed in uncoated fused-silica capillaries using a pH 9.5 ammonium acetate background electrolyte and normal polarity. MS detection of EtG and EtS occurred after negative ionization using a spray liquid containing 0.5%?v/v ammonia in isopropanol/water (60:40%, v/v). CZE-MS and CZE-MS2 results obtained after injection of solid-phase extracts for EtG and EtS and of diluted urine confirmed the presence of EtG and EtS in samples whose levels were previously determined by CZE with indirect UV detection. Detection limits of each compound were estimated to be around 2.0 (injection of diluted urine) and 0.2?μg/mL (extracts).     

Determination of acetic acid in aqueous samples, by water-phase derivatisation, solid-phase microextraction and gas chromatography

The direct derivatisation of acetic acid with n-hexyl chloroformate and with benzyl bromide in water was evaluated. With n-hexyl chloroformate, acetic acid did not give the n-hexyl acetate derivative, but the reaction of acetic acid with benzyl bromide in aqueous solution resulted in the formation of benzyl acetate. The derivatisation of acetic acid with benzyl bromide and the headspace solid-phase microextraction (SPME) of benzyl acetate were optimised. Under optimum conditions, the limit of detection for acetic acid was 260 nM, and the relative standard deviation of the overall procedure at 1.10(-4) M acetic acid was 15.6% (n = 10). A linear response was obtained in the 1 x 10(-4) to 5 x 10(-6) M concentration range (R2 = 0.993, n = 6). Although Carbowax-divinylbenzene (CW-DVB)-coated fibres exhibited a higher extraction capacity for benzyl acetate, polyacrylate (PA) was selected, because its mechanical stability was better than that of CW-DVB fibres. Moreover, the relative standard deviation of the SPME was better with PA (1.5%, n = 10 at 1 x 10(-5) M) than with CW-DVB-coated fibres (8.0%, n = 10 at 1 x 10(-5) M). Thus, a new analytical method for the quantitative determination of micromolar concentrations of acetic acid in the aqueous phase was developed. This method is based on water-phase derivatisation with benzyl bromide, headspace SPME with PA fibres and GC-FID. It was observed experimentally that benzyl alcohol formed by hydrolysis of the reagent affected the fibre-gas phase partitioning of benzyl acetate.     

Spectrofluorimetric determination of anthranilic acid derivatives based on terbium sensitized fluorescence.

Terbium sensitized fluorescence was used to develop a sensitive and simple spectrofluorimetric method for the determination of the anthranilic acid derivatives furosemide and mefenamic and tolfenamic acids. The method makes use of radiative energy transfer from anthranilates to terbium ions in alkaline methanolic solutions. Optimum conditions for the formation of the anthranilate-Tb3+ complexes were investigated. Under optimized conditions, the detection limits are 6 x 10(-9), 1.4 x 10(-8) and 9.0 x 10(-9) mol l-1 for furosemide, mefenamic acids and tolfenamic acid, respectively. The range of application is 2.5 x 10(-8)-5.0 x 10(-5) mol l-1 for all three drugs. The method was successfully applied to the determination of furosemide and mefenamic and tolfenamic acids in serum after extraction of the samples with ethyl acetate, evaporation of the organic layer under a stream of nitrogen at 40 degrees C and reconstitution of the residue with alkaline methanolic terbium solution prior to instrumental measurement. The mean recoveries from serum samples spiked with furosemide (5.0 x 10(-7), 2.0 x 10(-6) and 8.0 x 10(-6) mol l-1), mefenamic acid (3.0 x 10(-6), 9.0 x 10(-6) and 3.0 x 10(-5) mol l-1) and tolfenamic acid (3.1 x 10(-6), 12.5 x 10(-6) and 2.5 x 10(-5) mol l-1) were 96 +/- 8, 101 +/- 5 and 98 +/- 7%, respectively. The within-run precision (RSD) for the method for two serum samples of each drug varied from 2 to 8% and the day-to-day precision for two concentration levels varied from 2 to 13%.     

Forensic confirmatory analysis of ethyl sulfate—A new marker for alcohol consumption—by liquid-chromatography/electrospray ionization/tandem mass spectrometry

Ethyl sulfate (EtS)--a new direct marker for ethanol intake besides ethyl glucuronide (EtG) and others--was detected in urine samples by electrospray ionization tandem mass-spectrometry (LC-ESI-MS/MS). Ethyl sulfate sodium salt was used for method development, yielding a precursor [M - H]- m/z 125 and product ions m/z 97 [HSO4]- and m/z 80 [SO3]-. Pentadeuterated EtS (D5-EtS) was synthesized by esterification of sulfuric acid with anhydrous hexadeutero ethanol ([M - H]- m/z 130, product ions m/z 98 [DSO4]- and m/z 80 [SO3]-). After addition of D5-EtS and D5-EtG, urine samples were analyzed by direct injection into the gradient LC-MS/MS system. Analysis was performed in accordance with forensic guidelines for confirmatory analysis using one precursor and two product ions. EtS has been detected (in addition to EtG) in the urine samples of nine volunteers after drinking sparkling wine containing between 9 and 49 g of ethanol. Both EtS and EtG could be detected up to 36 h after consumption of alcohol. The excretion profile was found to be similar to that of EtG. No EtS was found in teetotalers' urine samples. Method validation parameters are presented. EtS was stable in urine upon storage up to twenty days at room temperature. In addition to EtG, EtS can be used to detect recent alcohol consumption, thus providing a second marker for the time range of up to approximately one day after elimination of ethanol from urine samples. The determination of EtS can be used in addition to EtG as proof of ethanol consumption in workplace monitoring programs.     

Determination of ethyl-glucuronide in hair for heavy drinking detection using liquid chromatography-tandem mass spectrometry following solid-phase extraction

The detection of ethyl-β-d-6-glucuronide (EtG), a stable phase II metabolite of ethanol, is of interest in both clinical and forensic contexts with the aim of monitoring alcohol abuse. We present a liquid chromatography-electrospray ionisation-tandem mass spectrometry method for the detection and quantification of EtG in hair. Thirty milligrams of washed and cut hair were cleaned up using solid-phase extraction graphite cartridges. Separation was then performed using an Uptisphere-3SI column, and the detection was operated in the negative mode. After validation, the method was applied to hair samples taken from four fatalities (F) with documented excessive drinking habits, 12 heavy drinkers (HD) and seven social drinkers (SD). The method exhibits limits of detection and quantification of 4 and 10 pg/mg, respectively. Intra- and inter-assay standard deviation and relative bias were less than 20% over the calibrating range (10 to 3,000 pg/mg). EtG hair concentrations in SD were <10 pg/mg and >50 pg/mg for F and HD (range, 54 to 497 pg/mg). The present assay appears convenient to carry out owing to the very small quantity of hair samples required to determine an effective heavy alcohol consumption (EtG hair concentration >50 pg/mg).     

A novel isomerization on interaction of antitumor-active azole-bridged dinuclear platinum(II) complexes with 9-ethylguanine. Platinum(II) atom migration from N2 to N3 on 1,2,3-triazole

The reactions of the dinuclear platinum(II) complexes, [[cis-Pt(NH(3))(2)](2)(mu-OH)(mu-pz)](NO(3))(2) (1, pz = pyrazolate), [[cis-Pt(NH(3))(2)](2)(mu-OH)(mu-1,2,3-ta-N1,N2)](NO(3))(2) (2, 1,2,3-ta = 1,2,3-triazolate), and a newly prepared [[cis-Pt(NH(3))(2)](2)(mu-OH)(mu-4-phe-1,2,3-ta-N1,N2)](NO(3))(2) (3, 4-phe-1,2,3-ta = 4-phenyl-1,2,3-triazolate), whose crystal structure was determined, with 9-ethylguanine (9EtG) have been monitored in aqueous solution at 310 K by means of (1)H NMR spectroscopy. The dinuclear platinum(II) complexes 1-3 each react with 9EtG in a bifunctional way to form 1:2 complexes, [[cis-Pt(NH(3))(2)(9EtG-N7)](2)(mu-pz)](3+) (4), [[cis-Pt(NH(3))(2)(9EtG-N7)](2)(mu-1,2,3-ta-N1,N3)](3+) (5), and [[cis-Pt(NH(3))(2)(9EtG-N7)](2)(mu-4-phe-1,2,3-ta-N1,N3)](3+) (6). The reactions of 2 and 3 involve a novel isomerization, in which the Pt atom, initially bound to N2 on the 1,2,3-ta, migrates to N3 after the first substitution by N7 of 9EtG. This isomerization reaction has been unambiguously characterized by 1D and 2D NMR spectroscopy and pH titration. The reactions of 2 and 3 with 9EtG show faster kinetics, and the second-order rate constants (k) for the reactions of 1-3are 1.57 x 10(-4), 2.53 x 10(-4), and 2.56 x 10(-4) M(-1) s(-1), respectively. The pK(a) values at the N1H site of 9EtG were determined for 4-6 from the pH titration curves. Cytotoxicity assays of 1-3 were performed in L1210 murine leukemia cell lines, respectively sensitive and resistant to cisplatin. In the parent cell line, 2 and 3 exhibit higher cytotoxicity compared to cisplatin, especially, 2 is 10 times as active as cisplatin. 1 was found to be less cytotoxic than cisplatin, but still in the active range and more active than cisplatin in a cisplatin-resistant cell line.     

Determination of ethyl sulfate in human serum and urine by capillary zone electrophoresis

The use of capillary zone electrophoresis (CZE) with indirect absorbance detection for the analysis of ethyl sulfate (EtS) in serum and urine was investigated. EtS is a direct metabolite of ethanol employed as marker for recent alcohol consumption. Fused-silica capillaries of 60 cm total length were either coated with cetyltrimethylammonium bromide (CTAB, 50 microm I.D. capillary) or poly(diallyldimethylammonium chloride) (PDADMAC, 100 microm I.D. capillary) to allow CZE analyses to be performed with reversed polarity. At pH 2.2 with a maleic acid/phthalic acid background electrolyte, both approaches provided reliable EtS serum levels down to 0.2 mg L(-1) (1.6 microM) for the analysis of solid-phase extracts that were prepared after chloride precipitation. Analysis of urines diluted to a conductivity of 5 S m(-1) and analyzed in the two capillary formats resulted in limits of quantification (LOQs) of 2 and 1 mg L(-1), respectively. With urines adjusted to 10 S m(-1) via dilution or condensation, an LOQ of 0.6 mg L(-1) (4.8 microM) was obtained in the CTAB coated capillary whereas in the PDADMAC-coated capillary of equal length not all matrix components were resolved from EtS. The developed assays are robust and suitable to monitor EtS in samples of individuals who consumed as little as one standard drink of an alcoholic beverage containing about 14 g of ethanol.     

Flash photolytic generation of ortho-quinone methide in aqueous solution and study of its chemistry in that medium

Flash photolysis of o-hydroxybenzyl alcohol, o-hydroxybenzyl p-cyanophenyl ether, and (o-hydroxybenzyl)trimethylammonium iodide in aqueous perchloric acid and sodium hydroxide solutions, and in acetic acid and biphosphate ion buffers, produced o-quinone methide as a short-lived transient species that underwent hydration back to benzyl alcohol in hydrogen-ion catalyzed (k(H+) = 8.4 x 10(5) M(-1) s(-1)) and hydroxide-ion catalyzed (k(HO)- = 3.0 x 10(4) M(-1) s(-1)) reactions as well as an uncatalyzed (k(UC) = 2.6 x 10(2) s(-1)) process. The hydrogen-ion catalyzed reaction gave the solvent isotope effect k(H+)/k(D)+ = 0.42, whose inverse nature indicates that this process occurs by rapid and reversible equilibrium protonation of the carbonyl oxygen atom of the quinone methide, followed by rate-determining capture of the carbocation so produced by water. The magnitude of the rate constant of the uncatalyzed reaction, on the other hand, indicates that this process occurs by simple nucleophilic addition of water to the methylene group of the quinone methide. Decay of the quinone methide is also accelerated by acetic acid buffers through both acid- and base-catalyzed pathways, and quantitative analysis of the reaction products formed in these solutions shows that this acceleration is caused by nucleophilic reactions of acetate ion rather than by acetate ion assisted hydration. Bromide and thiocyanate ions also accelerate decay of the quinone methide through both hydrogen-ion catalyzed and uncatalyzed pathways, and the inverse nature of solvent isotope effects on the hydrogen-ion catalyzed reactions shows that these reactions also occur by rapid equilibrium protonation of the quinone methide carbonyl oxygen followed by rate-determining nucleophilic capture of the ensuing carbocation. Assignment of an encounter-controlled value to the rate constant for the rate-determining step of the thiocyanate reaction leads to pK(a) = -1.7 for the acidity constant of the carbonyl-protonated quinone methide.     

Electrochemical adsorptive behavior of some fluoroquinolones at carbon paste electrode.

Cyclic voltammetry and differential pulse voltammetry were used to explore the adsorption behavior of three antibacterial agents at a carbon paste electrode. The drugs were accumulated on a carbon paste electrode, and a well-defined oxidation peak was obtained in acetate buffer (pH 5.0). The adsorptive stripping response was evaluated as a function of some variables such as the scan rate, pH and accumulation time. A simple, precise, inexpensive and sensitive voltammetric method has been developed for the determination of the cited drugs (Lomefloxacin (LFX), Sparfloxacin hydrochloride (SFX), and Gatifloxacin (GFX)). A linear calibration was obtained from 2 x 10(-7) M to 4 x 10(-5) M for LFX, 2 x 10(-7) M to 6 x 10(-5) M for SFX, and GFX. The limits of detection (LOD) were 4.2 x 10(-7), 7 x 10(-7) and 6.6 x 10(-7) M, while the limits of quantification (LOQ) were 1.4 x 10(-6), 2.3 x 10(-6) and 2.2 x 10(-6) M for LFX, SFX, and GFX, respectively. The R. S. D. of five measurements at the 1 x 10(-6) M level were 0.4, 0.5 and 0.3 for LFX, SFX and GFX, respectively. The method was applied to the determination of LFX, SFX and GFX in dilute urine samples and dosage forms, and compared with the HPLC method.     

Differential pulse polarographic study of the hydrolysis of endosulfan and endosulfan sulphate in emulsified medium. Application to the determination of binary mixtures of organochlorine pesticides

Hydrolysis reactions of endosulfan and endosulfan sulphate in the emulsified medium formed with ethyl acetate and a mixture of the two surfactants Hyamine 2389 and Triton X-405 are studied by differential pulse polarography. Besides the heptachlor-endosulfan sulphate pair, whose peak potentials are sufficiently different at pH 8.0 to allow their simultaneous determination, the organochlorine pesticide binary mixtures endosulfan-endosulfan sulphate, dieldrin-endosulfan and dieldrin-endosulfan sulphate can be determined based on their hydrolysis reactions in basic medium and on their different reaction rates. The endosulfan-endosulfan sulphate pair can be determined by allowing the mixture to hydrolyse at pH 11.0 and measuring the endosulfandiol peak for the determination of endosulfan. The analysis of the mixture dieldrin-endosulfan is based on endosulfan hydrolysis at pH 12.0 in which dieldrin is not hydrolysed. Dieldrin and endosulfan sulphate can also be determined simultaneously in a 0.5M sodium hydroxide medium. When determining one pesticide in binary mixtures containing a 5.0 x 10(-6)M concentration of the other pesticide, the lower limits of the calibrations obtained were: endosulfan-endosulfan sulphate mixture, 4.0 x 10(-6) and 1.0 x 10(-6)M respectively; heptachlor-endosulfan sulphate mixture, 2.0 x 10(-6)M for endosulfan sulphate; all other cases, 3.0 x 10(-6)M.     

Analysis of 5-methyltetrahydrofolate in human blood, serum and urine by on-line coupling of capillary isotachophoresis and zone electrophoresis

The predominant circulating folate coenzyme in plasma/serum, 5‐methyltetrahydrofolate (5‐MTHF) was determined in human blood, serum and urine using a method based on the hyphenation of capillary ITP and zone electrophoresis. Measurements were done with a commercially available instrument for capillary isotachophoresis equipped with a column‐switching system. The choice of electrolytes was limited by the instability of 5‐MTHF and volatility of electrolytes for the potential coupling of the instrumentation with MS detector. To get an insight into the separability of individual sample components in an isotachophoretic analysis, we constructed zone existence diagrams for isotachophoretic electrolyte systems having a leading electrolyte composed of acetate and ammonium of pH 4.5 and 7.0, hydrocarbonate and ammonium, pH 7.8, chloride and ammonium, pH 5.6, and chloride and creatinine, pH 5.0, with hydroxide ion as the terminator. For isotachophoretic preseparation, the non‐volatile leading electrolyte with good buffering capacity composed of 1×10⁻² M HCl and 2.5×10⁻² M creatinine, pH 5.0, and terminating electrolyte composed of 1×10⁻² M MES was selected as the most suitable. The optimum BGE for CZE analysis from the standpoint of analyte stability, separability and volatility for MS coupling was 1×10⁻² M acetate with 3.5×10⁻² M ammonium, pH 4.5. Using this combination of electrolytes, LODs reached with optical detection at 220 nm were 1.6×10⁻⁷ M in human blood, 1.1×10⁻⁷ M in human serum and 4.7×10⁻⁶ M in human urine. Estimated content of 5‐MTHF in blood and serum samples of women following oral daily administration of 0.8 mg of folic acid was 1.2×10⁻⁵ and 5.8×10⁻⁶ M, respectively.     

Quantitative investigation of the interaction between granulocyte-macrophage colony-stimulating factor and heparin by capillary zone electrophoresis

The interactions between granulocyte-macrophage colony-stimulating factor (GM-CSF) and heparin or low-molecular weight heparin (LMWH) were studied by CZE. It was found that GM-CSF could bind to both heparin and LMWH. The binding constants were calculated from Scatchard regression to be (6.5 +/- 0.8) x 10(5)/M and (11.2 +/- 0.7) x 10(5)/M, respectively. The specificity of the interaction between GM-CSF and heparin was also studied by employing another sulfated K carrageenan oligosaccharide as a control. Results showed that K carrageenan oligosaccharide could not interact with GM-CSF, indicating that GM-CSF could specifically interact with heparin.     

Determination of selenium(IV) and tellurium(IV) by differential pulse polarography

Differential pulse polarographic methods for the determination of selenium(IV) and tellurium(IV) in nitric acid medium are described. The peak current is maximal when 0.25M nitric acid medium is used, the DPP peaks for Se(IV) and Te(IV) being at -0.54 and -0.8 V vs. Ag/AgCl respectively. The peak current is a linear function of selenium concentration over three ranges, 5.1 x 10(-6)-1.3 x 10(-5), 1.27 x 10(-5)-1.27 x 10(-4) and 1.27 x 10(-4)-7.60 x 10(-4)M Se(IV), with different slopes. The plot for Te(IV) is linear over the range 0.78 x 10(-6)-9.40 x 10(-5)M.     

Determination of (E)-10-hydroxy-2-decenoic acid content in pure royal jelly: a comparison between a new CZE method and HPLC

A new CZE method was developed and compared with HPLC for the determination of (E)-10-hydroxy-2-decenoic acid (10-HDA) in royal jelly (RJ) samples of different geographical origin. The results obtained with the CZE method were highly correlated with those of HPLC (p < 0.01). Under optimized conditions, CZE employed minimal amounts of 50 mM tetraborate buffer as BGE, without the addition of organic solvents, EOF or pH modifiers. The CZE method showed a wide linear response range (0.006-0.808 mg 10-HDA/mL), a good sensitivity (LOD and LOQ were 0.002 and 0.004 mg/mL, respectively) and a satisfactory instrumental repeatability with respect to migration time and peak area (RSD% less than 1.0 and 2.0% on migration time for intra- and interday assay, respectively and less than 2.0 and for 4.0% on peak area for intra- and interday assay, respectively). The 10-HDA content in RJ ranged from 0.8 to 3.2 g/100 g of RJ and a significant difference (p < 0.05) was found between the Italian and extra-European average values: 2.5 and 1.6 g/100 g of RJ, respectively, according to the CZE data. The possibility of application of CZE for routine analyses on RJ and RJ based products to verify their authenticity is highlighted here.     

Simultaneous determination of ascorbic acid and acetylsalicylic acid in pharmaceutical formulations

A direct, simple, and practical first-derivative spectrophotometric method is described for simultaneous determination of ascorbic acid and acetylsalicylic acid. The effects of the solvent, excipients, and spectral variables on the analytical signal were investigated. The drugs were determined simultaneously with a 0.01 M methanolic hydrochloric acid solution as the solvent, and the signals were evaluated directly by using the zero-crossing method at 245.0 and 256.0 nm for acetylsalicylic acid and ascorbic acid, respectively. The method allows the simultaneous determinations of acetylsalicylic acid and ascorbic acid in the ranges of 6.6 x 10(-6) to 1.5 x 10(-4)M and 3.4 x 10(-6) to 2.0 x 10(-4)M, respectively, with standard deviation of <2.0%. The proposed method was applied to determinations of these drugs in tablets.     

Impact of hair-care products on FAEE hair concentrations in substance abuse monitoring

Previous studies have indicated that the use of high-ethanol-content (>65%) hair-care products may elevate fatty acid ethyl ester (FAEE) concentrations in hair. In this case series, nine individuals were identified by FAEE analysis to be chronic alcohol abusers in the context of child-welfare substance abuse monitoring. Based on patient claims of moderate or no alcohol consumption, the presence of ethanol in the patients’ hair-care regimens was investigated. Samples were additionally tested for the presence of ethyl glucuronide (EtG). From a total of nine patients, 12 hair samples were submitted for analysis. Patient histories were obtained as well as Material Safety Data Sheets (MSDS) listing hair-care product ethanol content. Hair samples were pre-washed to remove external contamination and analyzed for FAEE and EtG by GC-MS. According to the Society of Hair Testing consensus guidelines, FAEE levels exceeding 0.50 ng/mg and/or EtG levels exceeding 30 pg/mg indicate chronic excessive alcohol consumption. Upon initial analysis, the nine samples exhibited positive FAEE findings ranging from 0.496 to 4.984 ng/mg. MSDS review revealed the presence of ethanol from 10% to 95% by volume in at least one hair-care product used by each individual. Results of the EtG analysis ranged from 1.9 to 23.5 pg/mg. These findings indicate that regular use of products with ethanol content as low as 10% can impact FAEE results. EtG analysis should be used to confirm FAEE findings and appears to be unaffected by hair-care products, likely due to alternative mechanisms of incorporation.     

Capillary zone electrophoresis with a dynamic double coating for analysis of carbohydrate-deficient transferrin in human serum. Precision performance and pattern recognition

Capillary zone electrophoresis (CZE) with a dynamic double coating permits the simultaneous, individual, quantitative determination of transferrin (Tf) isoforms in human serum and thus carbohydrate-deficient transferrin (CDT), the most specific marker available today for the detection of chronic, excessive alcohol intake. CZE of serum Tf was carefully evaluated using the P/ACE MDQ with fused-silica capillaries of 50 microm I.D. and 60.2 cm total length, the CEofix CDT kit and the instrumental conditions recommended by the kit manufacturer. The precision performance assessed over a 20-day period according to the internationally accepted NCCLS EP5-A guidelines revealed the CZE assay as being highly reproducible with within-run and total precision being dependent on the Tf isoform level and RSD values ranging between 2.2 and 17.6%. Inter-day RSD values for asialo-Tf were noted to be between 9.8 and 11.5% and for disialo-Tf between 3.8 and 8.6%, whereas those for CDT levels of 0.87 and 4.31% of total Tf were determined to be 8.6 and 3.4%, respectively. The RSD values for trisialo-Tf, tetrasialo-Tf, pentasialo-Tf and hexasialo-Tf were found to be between 0.4 and 4.1%. Tf patterns are recognized and identified via detection times of Tf isoforms (intra-day and inter-day RSD values < 1.0% and < 1.7%, respectively), immunosubtraction of Tf and enzymatic sequential cleavage of sialic acid residues. Furthermore, heterozygous Tf BC and Tf CD variants are assigned via spiking with a known mixture of Tf isoforms (e.g. the serum of a healthy Tf C homozygote). Among the non-Tf peaks monitored, the CRP peak detected shortly before disialo-Tf was identified by immunosubtraction and peak magnitudes were found to correlate well with immunochemically determined CRP serum levels. The CZE assay with dynamic double coating could thereby be shown to be sensitive enough to determine elevated CRP levels in human serum. Furthermore, unusual peaks in the gamma-region were identified by customary serum protein CZE, immunosubtraction CZE and immunofixation.