Determination of cocaine and benzoylecgonine by direct injection of human urine into a column-switching liquid chromatography system with diode-array detection.

A method for the determination of cocaine (COC) and benzoylecgonine (BZE) in human urine using a column-switching liquid chromatography system is reported. A homemade precolumn (20 mm x 4.6 mm i.d.) dry-packed with Alltech ODS-C18 (35-750 microm) was employed as an extraction precolumn in order to extract and concentrate the COC and BZE from the human urine sample. The analytes were continuously transferred to the analytical column (Spherisorb-C8, 250 mm x 4.6 mm i.d.; dp = 5 microm) by means of the switching arrangement in the backflush mode. Detection was carried out at 235 nm in a UV-diode array detector. The validation of the method revealed analytes quantitative recoveries (96-102%) at three concentrations in the range from 0.25 to 4.00 and from 0.5 to 12.0 microg/mL for COC and BZE, respectively. These values demonstrate the excellent extraction efficiency of the precolumn. The detection limits for COC and BZE at a signal-to-noise ratio of 3 were 0.08 and 0.15 microg/mL when a sample volume of 50 microL was injected. The overlap of sample preparation, analysis and recondition of the precolumn increases the sample throughput to four samples per hour. The proposed method has been applied to the determination of COC and BZE in human urine samples from 73 suspecting drug addicts. Urine concentrations of 1.0-118.10 microg of BZE/mL and 0.1-41.0 microg of COC/mL were found.

HPLC determination of Vitamin D(3) and its metabolite in human plasma with on-line sample cleanup.

A HPLC method with automated column switching and UV-diode array detection is described for the simultaneous determination of Vitamin D(3) and 25-hydroxyvitamin D(3) (25-OH-D(3)) in a sample of human plasma. The system uses a BioTrap precolumn for the on-line sample cleanup. A sample of 1ml of human plasma was treated with 2ml of a mixture of ethanol-acetonitrile (2:1 (v/v)). Following centrifugation, the supernatant was evaporated to dryness under a stream of dry and pure nitrogen. The residue was reconstituted in 250muL of a solution of methanol 5mmoll(-1) phosphate buffer, pH 6.5 (4:1 (v/v)), and a 200mul aliquot of this solution was injected onto the BioTrap precolumn. After washing during 5min with a mobile phase constituted by a solution of 6% acetonitrile in 5mmoll(-1) phosphate buffer, pH 6.5 (extraction mobile phase), the retained analytes were then transferred to the analytical column in the backflush mode. The analytical separation was then performed by reverse-phase chromatography in the gradient elution mode with the solvents A and B (Solvent A: acetonitrile-phosphate buffer 5mmoll(-1), pH 6.5; 20:80 (v/v); solvent B: methanol-acetonitrile-tetrahydrofuran, 65:20:15 (v/v)). The compounds of interest were detected at 265nm. The method was linear in the range 3.0-32.0ngml(-1) with a limit of quantification of 3.0ngml(-1). Quantitative recoveries from spiked plasma samples were between 91.0 and 98.0%. In all cases, the coefficient of variation (CV) of the intra-day and inter-day-assay precision was

Determination of paraquat in human blood plasma using reversed-phase ion-pair high-performance liquid chromatography with direct sample injection.

This report describes the determination of paraquat (PQ) in human blood plasma samples by a direct-injection reversed-phase ion-pair chromatographic method. Blood plasma filtrate was injected directly into the LiChrospher(R) RP-18 alkyl-diol silica (ADS) precolumn integrated in a column switching system using a mixture of 3% 2-propanol and 10 mM sodium octane sulfonate (SOS) in a 0.05 M phosphate buffer (pH 2.8). After washing with this phase, the ADS precolumn was back-flushed with the analytical mobile phase consisting of 40% of methanol and 10 mM SOS in a 0.05 M phosphate buffer (pH 2.8) at a flow rate of 1.0 ml min(-1), in order to carry the analyte to a conventional reversed-phase analytical column, where the separation of PQ was achieved and finally detected by UV at 258 nm. The recoveries of PQ from human blood plasma samples ranged between 95.0 and 99.5% at nine different concentrations (from 0.05 to 3.00 microg of PQ ml(-1)) with coefficients of variation <2.5% (n=3). The precision expressed as relative standard deviation was below 3.5% for between-day and below 4.3% for within-day measurements (n=5). The detection limit (signal-to-noise ratio, S/N>3) was 0.005 microg ml(-1) with an injection volume of 200 microl. The proposed method is promising for the identification and quantification of PQ at low concentration levels and is suitable for its analysis in human blood plasma samples from intentional or accidental poisonings cases with a sample throughput of 5 samples per hour.

Flow analysis-hydride generation-Fourier transform infrared spectrometric determination of antimony in pharmaceuticals.

In this work, a flow analysis system with hydride generation and Fourier transform infrared (FTIR) spectrometric detection has been developed for the determination of antimony in pharmaceuticals. The method is based on the on-line mineralization/oxidation of the organic antimonials present in the sample and pre-reduction of Sb(V) to Sb(III) with K(2)S(2)O(8) and KI, respectively; prior to the stibine generation. The gaseous SbH(3) is separated from the solution in a gas phase separator, and transported by means of a nitrogen carrier into a short pathway (10 cm) IR gas cell, where the corresponding FTIR spectrum is acquired by accumulating 3 scans in a continuous mode. The 1893 cm(-1) band was used for the quantification of the antimony. The procedure is carried out in a closed system, which reduces sample handling and makes possible the complete automation of the antimony determination. The figures of merit of the proposed method (linear range: 0-600 mg l(-1), limit of detection (3sigma)=0.9 mg l(-1), limit of quantification (10sigma)=3 mg Sb l(-1), precision (R.S.D.) less than 1% and sample frequency=28 h(-1)), are appropriate for the designed application. Furthermore, precise and accurate results were found for the analysis of different antimonial pharmaceutical samples, indicating that the methodology developed represents a valid alternative for the determination of antimony in pharmaceuticals, which could be suitable for the routine control analysis.

Column-switching high-performance liquid chromatographic analysis of carbamazepine and its principal metabolite in human plasma with direct sample injection using an alkyl-diol silica (ADS) precolumn.

In this paper, the on-line coupling of solid-phase extraction, based on a restricted-access support with high-performance reverse phase chromatography for the analysis of carbamazepine (CBZ) and carbamazepine-10,11-epoxide (CBZ-E) in human plasma samples is described. A precolumn packed with 25 mum C(18) alkyl-diol support is used for direct plasma injection. Using column-switching techniques, the analytes were enriched on the precolumn by a 5 mM phosphate buffer (pH 7) with 2% of methanol solution at a flow-rate of 0.8 ml min(-1), while proteins and endogenous hydrophilic substances in plasma were washed off to waste. The enriched analytes were then back-flushed onto the analytical C(18) column, separated by a mixture of 10 mM phosphate buffer (pH 7) acetonitrile (70:30 v/v) solution at a flow-rate of 1.0 ml min(-1) and detected by the ultraviolet absorbance set at 212 and 285 nm and without transfer loss. Linear calibration graphs were obtained for sample injection volumes of 50 (0.2-4.0 of mug of CBZ ml(-1) and 0.1-5.0 mug of CBZ-E ml(-1), respectively), and 20 mul (5.0-20.0 mug of CBZ ml(-1)); in either case the r-value was >0.9963. Recoveries from spiked plasma samples were quantitative for both analytes and the coefficients of variation were below 3.83%. The lowest samples concentrations that can be quantified with acceptable accuracy and precision was 0.2 mug CBZ ml(-1) and 0.1 mug CBZ-E ml(-1) when a sample volume of 50 mul was injected. Concentrations of 0.08 and 0.05 mug ml(-1) of CBZ and CBZ-E were considered the limit of detection for a signal-to-noise ratio of 3. Furthermore, the developed column-switching method was successfully applied to the determination of CBZ and CBZ-E in plasma samples of patients submitted to CBZ therapy.

Sequential determination of Se(IV) and Se(VI) by flow injection-hydride generation-atomic absorption spectrometry with HCl/HBr microwave aided pre-reduction of Se(VI) to Se(IV).

In this study a flow injection (FI) system used in conjunction with hydride generation (HG), atomic absorption spectrometry (AAS) and microwave (MW) aided pre-reduction of selenite (Se(IV)) to selenate (Se(IV)) with HCl:HBr has been developed in order to differentiate both inorganic selenium species. As full control of the MW reduction step is possible, the experimental approach allows the use of milder acidic conditions (10% v/v of HCl and HBr) than those conventionally accomplished with hydrochloric acid alone (>/=50% v/v). Experimental parameters were optimized by the univariate optimization method. In either case, the linear range was from 1.0 to 30 mug l(-1). The detection limits based on 3sigma of the blank signal were 0.25 mug l(-1) for Se(IV) and 0.30 mug l(-1) for Se(VI). The reproducibility, about 3% RSD and recoveries of different amounts of Se(VI) and Se(IV) added to water and orange juice samples (97-103%) were good. The main advantage of the proposed method is that the sequential determination of Se(IV) and Se(VI) is performed at a high sampling frequency (ca. 50 samples per h) in a closed system without Se losses, and with a minimum sample waste, operator attention, and sample manipulation.

Serum trace elements and fat-soluble vitamins A and E in healthy pre-school children from a Venezuelan rural community.

Zn (SZn), Cu (SCu), Fe (SFe), vitamin A (SVA) and vitamin E (SVE) were measured in blood serum samples of 85 healthy pre-school children aged 2-6 yr. from the rural community of Canaguá, Mérida State, Venezuela. The relationship between these biochemical indexes was also investigated. The mean serum concentrations of zinc, copper, iron, vitamin A and vitamin E were 0.74 +/- 0.25, 1.18 +/- 0.30, 0.76 +/- 0.20, 0.30 +/- 0.15 and 5.87 +/- 0.43 mg/L, respectively. There was a tendency for SZn to increase with age, whereas SCu and SVA decreased. There was no significant difference in serum trace elements and fat-soluble vitamin concentration between males and females in the different age groups. SFe tended to be lower than that reported in the literature. However, the age groups studied showed no statistically significant sex- and age-related differences. The present study shows that there is a complex interaction between SZn, SCu, SFe, SVA, SVE and age of the children. Multiple regression analysis showed serum zinc was strongly related to serum copper, and serum iron. Serum vitamin A was strongly related to serum zinc and serum vitamin E, whereas serum vitamin E was strongly related to serum zinc, serum copper, and serum vitamin A. On the other hand, our observations also suggest that more detailed studies of these metals and fat-soluble vitamins should be carried out, and that the study should include nutritional surveys, metabolic balances and associations between SZn, SCu, SFe, SVA and SVE and anthropometric variables (height, weight, body mass index and skinfold thickness).

Determination of methylmercury and inorganic mercury in whole blood by headspace, cryofocusing gas chromatography and atomic absorption spectrometry.

A method for the determination of different mercury species in whole blood is described. Inorganic mercury (InHg) was determined in 2 ml of standard solutions or blood samples using head space (HS) injection coupled to atomic absorption spectrometry (AAS) after treatment with concentrated sulfuric and tin(II) chloride as a reductant agent in a closed HS vial. After stirring, the InHg was converted to elementary mercury and carried with a nitrogen flow through a quartz cell heated at 200 degrees C and the absorbance signal was evaluated by AAS. For the determination of methylmercury (MeHg), 2 ml of a standard solution or a blood sample were treated with 10 mg of iodoacetic acid and 0.4 ml of concentrated H2SO4. Then, the MeHg species were HS-injected into a gas chromatograph (GC), separated on a semicapillary column (AT-1000) with a flow of helium, then carried to the quartz cell heated at 1000 degrees C and detected by AAS. The high content of salts in blood samples, where sodium chloride is the major component (0.14 mol l-1), affected the gas-liquid distribution coefficient of both mercury species in the HS vial. A linear calibration graph was obtained in the ranges 1-20 and 1-125 micrograms Hg l-1 added as InHg and MeHg, respectively. The detection limits for InHg and MeHg were 0.6 and 0.2 microgram Hg l-1, respectively. The relative standard deviations for eleven independent measurements were 5% for both mercury species. Recovery values ranging from 98 to 106% for InHg and from 95 to 105% for MeHg and from 93 to 95% for ethylmercury (EtHg) were obtained. The accuracy of the proposed method was also established by the analysis of certified whole blood samples for InHg and MeHg. No difference between the sum of these two species determined by our procedure and the recommended total mercury concentrations in the certified samples was observed. Results for the determination of MeHg and InHg in 30 controls and 30 dentists are presented to illustrate the practical utility of the proposed method.

Determination of bismuth in biological samples using on-line flow-injection microwave-assisted mineralization and precipitation/dissolution for electrothermal atomic absorption spectrometry.

An on-line automated flow injection system with microwave-assisted sample digestion for the electrothermal atomic absorption spectrometric determination of bismuth in biological materials is described. After the exposure of the sample to microwave radiation, the analyte was subject to a precipitation/dissolution process. Bismuth was precipitated with the stannite ion in basic medium and collected on the walls of a knotted coil, while the other matrix components flowed downstream to waste. The precipitate was dissolved with nitric acid and a sub-sample was collected in a capillary of a sampling arm assembly, to introduce 20 mul volumes into the graphite tube by means of positive displacement with air through a time-based injector. The analytical figures of merit were first evaluated by filling the sampling arm with a standard solution of bismuth and thereafter injecting aliquots of this solution into the atomizer. The calibration graph was linear from the detection limit (8 pg) to 1.2 ng of bismuth. The sensitivity was of 26.8 mug l(-1) for 0.2 A-s and the characteristic mass (m(o)) was of 11.8 pg/0.0044 A-s. The precision of the method, evaluated by replicate analyses of solutions containing 20 and 200 pg of bismuth, were 5.5 and 3.0% (n=10), respectively. When solutions were introduced in the flow system here described, the calibration graph was linear in the range 0.04-6.0 mug l(-1), which means that a preconcentration factor of 10 was obtained for bismuth. The precision slightly deteriorated, e.g. the replicate analysis of solutions containing 1 and 10 pg of bismuth were 7.1 and 5.3% (n=10), respectively. However, the recoveries values obtained with urine and whole blood bismuth spiked samples were over 96.5% and the agreement between observed and certified values was good.

Head-space flow injection for the on-line determination of iodide in urine samples with chemiluminescence detection.

A simple head-space (HS) flow injection (FI) system with chemiluminescence (CL) detection for the determination of iodide as iodine in urine is presented. The iodide is converted to iodine by potassium dichromate under stirring in the closed HS vial, and the iodine is released from urine by thermostatting and is carried in a nitrogen flow through an iodide trapping solution. The concomitant introduction of aliquots of iodine, luminol and cobalt(II) solutions by means of a time-based injector into an FI system allowed its mixing in a flow-through cell in front of the detector. The emission intensity at 425 nm was recorded as a function of time. The salting-out of the standard solutions affected the gas-liquid distribution coefficient of iodine in the HS vial. The typical analytical working graphs obtained under the optimized experimental conditions were rectilinear from 0 to 5 mg l(-1) iodine, achieving a precision of 2.3 and a relative standard deviation of 1.8 for ten replicate analyses of 50 and 200 microg l(-1) iodine. However, a second-order process becomes significant at higher iodine concentrations (from 10 to 40 mg l(-1)). The detection limit of the method is 10 microg l(-1) (80 ng) iodine when 8 ml samples are taken. Data for the iodide content of 10 urine samples were in good agreement with those obtained by a conventional catalytic method, and recoveries varied between 101 and 103% for urine samples spiked with different amounts of iodide. The analysis of one sample takes less than 20 min. In the present study the iodide levels found for 100 subjects were 86.8 +/- 19.0 (61-125) microg l(-1), which is lower than the WHO's optimal level (150-300 microg per day).

A factorial design to determine the effects of some variables in the long-term stability of methylmercury standard solutions in water.

A factorial composite design has been employed for a rapid exploration of the effect of some parameters on the stability of spiked methylmercury iodide solutions. The analysis has been performed by capillary gas chromatography with electron-capture detection. The results obtained by the factorial and the univariate methods are in a good agreement. Although, the factorial design does not allow a discrimination of the significance among the same kind of factors, it greatly reduces the number of experiments when the different variables are investigated.

Gastric tissue selenium levels in healthy persons, cancer and non-cancer patients with different kinds of mucosal damage.

The gastric tissue levels of selenium (Se) were examined in 15 healthy subjects aged 18-43 (mean 36 +/- 12) years, 17 patients with stomach cancer aged 23-79 (mean 60 +/- 17) years, 20 patients with mild gastritis aged 18-65 (mean 35 +/- 12) years, 13 patients with chronic gastritis aged 28-69 (mean 47 +/- 15) years, 33 patients with erosive gastritis aged 16-70 (mean 49 +/- 17) years and 23 patients with ulcers aged 22-76 (mean 49 +/- 17) years. The Se concentration was determined by electrothermal atomic absorption spectrometry (ETAAS) with palladium as a matrix modifier. The mean +/- the standard deviation (SD) of the gastric tissue Se levels were 473 +/- 80, 36 +/- 9.0, 355 +/- 243, 567 +/- 246, 571 +/- 241 and 813 +/- 427 micrograms/Kg in healthy subjects and in patients with cancer, ulcers, mild gastritis, chronic gastritis and erosive gastritis, respectively. The Se concentration in the biopsies of patients with gastric ulceration and cancer were significantly lower than that in patients with gastritis (p < 0.05) and the other conditions (p < 0.0001).

Selective determination of antimony(III) and antimony(V) in liver tissue by microwave-assisted mineralization and hydride generation atomic absorption spectrometry.

Antimony(III) and antimony(V) species have been selectively determined in liver tissues by optimizing the acidic conditions for the evolution of stibine using the reduction with sodium borohydride. The results show that a response for Sb(III) of 0.5 to 20 microg l(-1) was selectively obtained from samples in a 1 mol l(-1) acetic acid medium. The best response for total antimony from 1 to 20 microg l(-1) is obtained after sample treatment with a 0.5 mol l(-1) sulfuric acid and 10% w/v potassium iodide. Microwave digestion has been necessary to release quantitatively antimony species from sample slurries. The amount of Sb(V) was calculated from the difference between the value for total antimony and Sb(III) concentrations. A relative standard deviation from 2.9 to 3.1% and a detection limit of 0.15 and 0.10 microg l(-1) for Sb(III) and total Sb has been obtained. The average accuracy exceeded 95% in all cases comparing the results obtained from recovery studies, electrothermal atomic absorption spectrometry and the analysis of certified reference materials.

Comparative study of different approaches for the flow injection-fourier transform infrared determination of toluene in gasolines.

A single channel flow injection manifold has been employed to carry out the direct determination of toluene in gasolines by FT-IR without any sample pretreatment and by using different strategies. Toluene can be directly determined by measuring the absorbance at 728 cm(-1), using a base line established between 835 and 575 cm(-1); and in this case a limit of detection of 0.01% (v/v) can be obtained with a dynamic range up to 2% (v/v). In some cases it could be convenient to determine toluene by derivative flow-injection FT-IR in order to avoid matrix interferences in the analysis of some types of gasolines. Carrying out the first order derivative FI-FT-IR measurements on the 728 cm(-1) band between the peak at 731 and the valley at 725 cm(-1) toluene can be determined without problems in establishing the base line and with a limit of detection of 0.01% (v/v). On the other hand, the use of the quotient between the first order derivative absorbance values of toluene, at the 728 cm(-1) band, and benzene, at the 675 cm(-1) band, can be applied for the determination of toluene in gasolines in which benzene has been previously analyzed, avoiding problems related to the determination of the base line and making measurements independent of the spacer employed.

Simultaneous determination of acetylsalicylic acid and caffeine in pharmaceuticals by flow injection with fourier transform infrared detection.

A fast quality control methodology has been developed for the simultaneous determination of acetylsalicylic acid (ASA) and caffeine in pharmaceuticals by flow injection-Fourier Transform Infrared Spectrometry. The method is based on the solubilization of ASA and caffeine in CH(2)Cl(2) and the use of a flow system to introduce samples and standards in the spectrometer. Two solutions, containing 90 and 110% of the reported concentration of the two active principles in the sample, were employed in order to control the extreme tolerance levels accepted by the International Pharmacopeia for the composition of formulations. A 300 mul volume of each solution was injected in turn, into a carrier stream of the sample, and their absorbances were measured at wavenumbers of 1770 and 1661/cm for ASA and caffeine, respectively. The absorbance values obtained for the carrier, at the same wavenumbers, were interpolated between those obtained for the standards. This rapid methodology provides a simultaneous quantitative evaluation of the concentration of ASA and caffeine in pharmaceuticals. The method permits us to carry out 53 injections/hr and provides a sensitivity of 0.231 absorbance units/mg ml/mm for ASA and 1.065 for calteine. Accurate results were obtained for the analysis of different samples containing one or both compounds in the presence of excipients which are insoluble in CH(2)Cl(2) and also in the presence of other active principles, such as acetaminophen or ergotamine tartrate.

FIA-FT-IR determination of ibuprofen in pharmaceuticals.

A method has been developed for the determination of Ibuprofen (2-[4-isobutylphenyl]-propionic acid) in pharmaceuticals by FT-IR, using the carbonyl band which this compound presents at 1710 cm(-1) in carbon tetrachloride solutions. Samples are dissolved in carbon tetrachloride. In this solvent the excipients are not soluble and so the drug can be directly determined without any additional treatment. The use of a simple FIA manifold permits one to carry out this analysis with a low consumption of reagent and the FT-IR provides a continuous monitoring of the spectral base-line which permits an accurate determination of the maximum in the absorbance band. Also, the FIA system permits easy and fast sampling and cleaning of the measurement cell. The method has a dynamic range between 0.5 and 20 mg/ml with a sensitivity of 0.366 +/- 0.004 au . mg(-1) . ml . mm(-1) and a variation coefficient of 0.8% for 5 independent measurements of a real sample containing 200 mg of Ibuprofen per capsule. The developed procedure provides concentration values comparable with those found by UV spectrophotometry in the analysis of real samples but is free from matrix interferences.

Blood serum selenium in the province of Mérida, Venezuela, related to sex, cancer incidence and soil selenium content.

Blood serum selenium levels were measured in healthy subjects of six districts located throughout the province of Mérida, Venezuela. There were either high (87-115 micrograms/L) or low (58-72 micrograms/L) serum selenium-containing districts. Serum selenium in males from three districts were statistically significantly lower (p less than 0.001) than in females. There was a general tendency for serum selenium to increase with age. On the average, a reduction in cancer was observed in the districts with high serum selenium content. Further, serum selenium was lower in male patients from the Mérida district with cancer disease. Association between selenium in soil and in the blood serum of healthy subjects has been observed.

A comparative study of methods for determining selenium in biological materials.

A comparative study of the analytical performance of fluorimetric spectrophotometric, atomic absorption spectrometric, flow injection analysis with atomic absorption spectrometric, flow injection analysis with atomic absorption spectrometric detection, hydride generation with atomic absorption spectrometric detection and hydride generation with molecular emission cavity analysis detection methods has been carried out for the determination of selenium in biological materials. Based on results concerning detection limit, linearity and sensitivity, only the fluorimetric and hydride generation with atomic absorption spectrometric detection methods were suitable for the determination of selenium in biological materials. Whereas, the spectrophotometric, flame absorption spectrometric flow injection-atomic absorption spectrometric and hydride generation with molecular emission cavity detection, due to its worse detection limits and poorer sensitivities, were found to be unsuitable for the determination of selenium in such matrices. The accuracy of the fluorimetric and hydride generation with atomic absorption spectrometric detection methods were tested by using NBS standard reference materials.

A comparative study of methods for determining selenium in biological materials

En el presente estudio se realiza una comparación experimental de las características analíticas de diferentes métodos utilizados para la determinación de selenio en materiales biológicos. Los métodos evaluados fueron: fluorimetría, espectrofotometría de absorción atómica, inyección en flujo continuo con detección por espectrofotometría de absorción atómíca, espectrofotometría y generación de hidruro con detección por espectrofotometría de absorción atómica y emisión molecular en cavidad. Basados en las detecciones límites, lineridades y sensitividades obtenidas para los diferentes métodos, permiten concluir que solamente los métodos de generación de hidruro con detección por espectrofotmetría de absorción atómica y el fluorimétrico pueden utilizarse para la determinación de selenio en materiales biológicos. La exactitud de estos dos métodos se evaluó determinado el contenido de selenio en estándares de la NBS

Direct determination of sodium and potassium in blood serum by flow injection and atomic absorption spectrophotometry.

A simple and reliable method for the measurement of sodium and potassium in blood serum without any sample dilution by using flow injection and atomic absorption spectrophotometry is described. A sample throughout of 100 measurements per hour is possible. Matrix problems have not been encountered. The coefficient of variation for within-run determinations is about 1,14 and 2,36% for sodium and potassium, respectively, in serum samples (n = 10). The method is easily adaptable to pediatric research, because of the low required sample volume of 5 microliter.