Analysis of Terbumeton and its Major Metabolites by SPE and DAD-HPLC in Soil Bulk Water

A rapid and inexpensive method for simultaneous quantification of terbumeton (TER), and its major potential metabolites (TED; terbumeton-desethyl, TOH; terbumeton-2-hydroxy and TID; terbumeton-deisopropyl) in soil bulk water (SBW) samples is proposed. The analytical method involves extraction–concentration from SBW samples using a graphitized carbon black (GCB) cartridge followed by their separation–detection by reversed-phase high-performance liquid chromatography analysis using a C₁₈ column and a diode array detector. A mobile phase of acetonitrile−0.005 mol L⁻¹ phosphate buffer (pH 7.0) (35:65, v/v) at a flow rate of 0.8 mL min⁻¹ in isocratic elution mode has been used. After optimization of the extraction and separation conditions, this method can be used for the simultaneous determination of investigated compounds in the range of the international limits of 0.1 μg L⁻¹. For TER the detection limit was 0.009 μg L⁻¹ and it was 0.100, 0.550, and 0.480 μg L⁻¹ for TED, TOH, and TID, respectively. The recoveries of TER, TED, TOH, and TID from SBW samples, measured at three levels of concentration range, were found to be between 48.0 and 102.0%. The intra-day precision measured by relative standard deviation (RSD) was always lower than 9.0%.     

Determination of lead in wine by hydride generation atomic fluorescence spectrometry in the presence of hexacyanoferrate(III)

A rapid, accurate, and precise method is described for the determination of Pb in wine using continuous-flow hydride generation atomic fluorescence spectrometry (CF-HGAFS). Sample pretreatment consists of ten-fold dilution of wine followed by direct plumbane generation in the presence of 0.1 mol L⁻¹ HCl and 1% m/v K₃[Fe(CN)₆] with 1% m/v NaBH₄ as reducing agent. An aqueous standard calibration curve is recommended for Pb quantification in wine sample. The method provides a limit of detection and a limit of quantification of 0.3 μg L⁻¹ and 1 μg L⁻¹, respectively. The relative standard deviation varies between 2–6% (within-run) and 4–11% (between-run) at 3–30 μg L⁻¹ Pb levels in wine. Good agreement has been demonstrated between results obtained by CF-HGAFS and direct electrothermal atomic absorption spectrometry in analyses of red and white wines within the concentration range of 9.2–25.8 μg L⁻¹ Pb.     

HPLC method for determination of cefuroxime in plasma

Summary This paper describes an HPLC method for the determination of cefuroxime in human plasma. The method uses solid phase extraction (SPE) and has acceptable sensitivity, precision and accuracy. The limit of quantification in plasma samples is 0.1 μg mL⁻¹. Calibration curves were linear within 0.1–20 μg mL⁻¹, with mean correlation coefficient of 0.9982. Mean inter day precision and accuracy were 7.8% and 6.4%, respectively. The method was applied to determine cefuroxime levels in patients receiving cefuroxime, 3 time per day.     

Multiresidue procedures for determination of triazine and organophosphorus pesticides in water by use of large-volume PTV injection in gas chromatography

Summary Two procedures, based on large-volume injection with a programmed-temperature vaporizer (PTV), have been developed for the determination of several triazine and organophosphorus pesticides. The use of PTV for injection in gas chromatography (GC) has enabled the introduction of up to 200 μL sample extract into the GC, thus increasing the sensitivity of the method. PTV injection has been combined off-line with two different microextraction procedures—liquid-liquid partition and solid-phase extraction. A simple and rapid off-line liquid-liquid microextraction procedure (5 mL water/1 mL methyltert-butyl ether) was applied to surface water samples spiked at levels between 0.01 and 5μg L⁻¹. Recoveries of the overall procedure were >80% and the precision was better than 15%. Detection limits were <30 ngL⁻¹ from 200-μL injections in GC-NPD analysis of triazines and GC-FPD analysis of organophosphorus pesticides. Off-line automated solid-phase extraction with C₁₈ cartridges has been applied to water samples (50 mL) spiked at 0.01, 0.1 and 1 μg L⁻¹. The overall procedure was satisfactory (recoveries >80% and coefficients of variation <12%) and the limits of detection ranged from 1 to 9 ng L⁻¹. Finally, several surface water samples were anlysed, and triazine herbicides were detected at concentrations of approx. 0.1–0.2 μg L⁻¹. The results were similar to those obtained by conventional solvent extraction then GC-MSD after splitless injection of 2 μL.     

Isocratic Reversed-Phase HPLC for Simultaneous Separation and Determination of Seven Antiepileptic Drugs and Two of their Active Metabolites in Human Plasma

A simple reversed-phase high-performance liquid chromatographic (HPLC) method has been developed for the simultaneous determination of the antiepileptic drugs (AEDs) zonisamide (ZNS), primidone (PRI), lamotrigine (LTG), phenobarbital (PB), phenytoin (PHT), oxcarbazepine (OXC), and carbamazepine (CBZ) and two of their active metabolites, monohydroxycarbamazepine (MHD) and carbamazepine 10,11-epoxide (CBZE) in human plasma. Plasma (100 μL) was pretreated by deproteinization with 300 μL methanol containing 20 μg mL⁻¹ propranolol hydrochloride as internal standard. HPLC was performed on a C₈ column (4.6 mm × 250 mm; particle size 5 μm) with methanol–acetonitrile–0.1% trifluoroacetic acid, 235:120:645 (v/v), as mobile phase at a flow rate of 1.5 mL min⁻¹. ZNS, OXC, and CBZ were monitored by UV detection at 235 nm, and PRI, LTG, MHD, PB, PHT, and CBZE by UV detection at 215 nm. Relationships between response and concentration were linear over the concentration ranges 1–80 μg mL⁻¹ for ZNS, 5–50 μg mL⁻¹ for PRI, 1–25 μg mL⁻¹ for LTG, 1–50 μg mL⁻¹ for MHD, 5–100 μg mL⁻¹ for PB, 1–10 μg mL⁻¹ for CBZE, 0.5–25 μg mL⁻¹ for OXC, 1–50 μg mL⁻¹ for PHT, and 1–25 μg mL⁻¹ for CBZ. Intra-day and inter-day reproducibility were adequate (coefficients of variation were ≤11.6%) and absolute recovery ranged from 95.2 ± 6.13 to 107.7 ± 7.76% for all the analytes; for the IS recovery was 98.69 ± 1.12%. The method was proved to be accurate, reproducible, convenient, and suitable for therapeutic monitoring of the nine analytes.     

Flow injection spectrofluorimetric determination of iron(III) in water using salicylic acid

A simple and fast flow injection fluorescence quenching method for the determination of iron in water has been developed. Fluorimetric determination is based on the measurement of the quenching effect of iron on salicylic acid fluorescence. An emission peak of salicylic acid in aqueous solution occurs at 409 nm with excitation at 299 nm. The carrier solution used was 2 × 10⁻⁶ mol L⁻¹ salicylic acid in 0.1 mol L⁻¹ NH₄⁺/NH₃ buffer solution at pH 8.5. Linear calibration was obtained for 5–100 μg L⁻¹ iron(III) and the relative standard deviation was 1.25 % (n = 5) for a 20 μL injection volume iron(III). The limit of detection was 0.3 μg L⁻¹ and the sampling rate was 60 h⁻¹. The effect of interferences from various metals and anions commonly present in water was also studied. The method was successfully applied to the determination of low levels of iron in real samples (river, sea, and spring waters).     

Determination of eleven priority EPA phenolics at ng L−1 levels by on-line solid-phase extraction and liquid chromatography with UV and electrochemical detection

Summary The eleven priority, EPA phenolic pollutants were determined by liquid chromatography followed by two detectors in series; UV and electrochemical. Three different adsorbents, Envi-Carb (a carbon black) and two functionalized polymeric resins, Bond Elut PPL and another synthesized in our laboratory with an ocarboxybenzoyl moiety, were compared for solid-phase extraction (SPE) to detect lower concentrations of the eleven phenolics in natural waters. Higher recoveries were obtained using the functionalized polymeric adsorbents compared with Envi-Carb. When real samples were analysed, the synthetic adsorbent gave lower interference than Bond Elut PPL and phenol was determined at low levels with no humic and fulvic acid inter-ference when Na₂SO₃ was added. The linearity range for most compounds in tap water was 0.05–20 μg L⁻¹ and the limits of detection were <35 ng L⁻¹. Repeatability and reproducibility between days for real samples spiked at 0.1 μg L⁻¹, expressed as relative standard deviation, were <8% and 10%, respectively.     

Simple and Rapid Determination of Benzoylphenylurea Pesticides in River Water and Vegetables by LC–ESI-MS

Liquid chromatography with electrospray mass spectrometry (LC–ESI-MS) instrumentation equipped with a single quadrupole mass filter has been used to determine several benzoylphenylurea insecticides (diflubenzuron, triflumuron, hexaflumuron, lufenuron and flufenoxuron). Chromatographic and MS parameters were optimised to obtain the best sensitivity and selectivity for all pesticides. Solid-phase extraction (SPE) using C₁₈ cartridges was applied for preconcentration of pesticide trace levels in river water samples. Recoveries of benzoylphenylurea pesticides from spiked river water (0.01 and 0.025 μg L⁻¹) were between 73 and 110% and detection limits were between 3.5 and 7.5 ng L⁻¹. The applicability of the method to the determination of benzoylphenylurea insecticides in spiked cucumber, green beans, tomatoes and aubergines was evaluated. Samples were extracted into dichloromethane without any clean-up step. The limits of detection ranged from 1.0 to 3.2 ng mL⁻¹ (0.68 and 2.13 μg kg⁻¹ in the vegetable samples). Mean recoveries ranged from 79 to 114% at spiking levels of 0.01 and 0.03 mg kg⁻¹. The method was applied to determine traces of benzoylphenylureas in both river water and vegetable samples with precision values lower than 10%. Interferences due to the matrix effect were overcome using matrix-matched standards.     

Determination of glyphosate and AMPA in surface and waste water using high-performance ion chromatography coupled to inductively coupled plasma dynamic reaction cell mass spectrometry (HPIC–ICP–DRC–MS)

A novel method employing high-performance cation chromatography in combination with inductively coupled plasma dynamic reaction cell mass spectrometry (ICP–DRC–MS) for the simultaneous determination of the herbicide glyphosate (N-phosphonomethylglycine) and its main metabolite aminomethyl phosphonic acid (AMPA) is presented. P was measured as ³¹P¹⁶O⁺ using oxygen as reaction gas. For monitoring the stringent target value of 0.1 μg L⁻¹ for glyphosate, applicable for drinking and surface water within the EU, a two-step enrichment procedure employing Chelex 100 and AG1-X8 resins was applied prior to HPIC–ICP–MS analysis. The presented approach was validated for surface water, revealing concentrations of 0.67 μg L⁻¹ glyphosate and 2.8 μg L⁻¹ AMPA in selected Austrian river water samples. Moreover, investigations at three waste water-treatment plants showed that elimination of the compounds at the present concentration levels was not straightforward. On the contrary, all investigated plant effluents showed significant amounts of both compounds. Concentration levels ranged from 0.5–2 μg L⁻¹ and 4–14 μg L⁻¹ for glyphosate and AMPA, respectively.     

Simultaneous RP-LC Determination of Losartan Potassium, Ramipril, and Hydrochlorothiazide in Pharmaceutical Preparations

A simple, rapid, and precise reversed-phase high-performance liquid chromatographic method has been developed for simultaneous determination of losartan potassium, ramipril, and hydrochlorothiazide. The three drugs were separated on a 150 mm × 4.6 mm i.d., 5 μm particle, Cosmosil C₁₈ column. The mobile phase was 0.025 m sodium perchlorate–acetonitrile, 62:38 (v/v), containing 0.1% heptanesulphonic acid, pH adjusted to 2.85 with orthophosphoric acid, at a flow rate of 1.0 mL min⁻¹. UV detection was performed at 215 nm. The method was validated for linearity, accuracy, precision, and limit of quantitation. Linearity, accuracy, and precision were acceptable in the ranges 35–65 μg mL⁻¹ for losartan, 1.75–3.25 μg mL⁻¹ for ramipril, and 8.75–16.25 μg mL⁻¹ for hydrochlorothiazide.     

Quantitative Analysis of Sulfonamide Residues in Natural Animal Casings by HPLC

A multiresidue method has been developed for the simultaneous determination of sulfadiazine, sulfathiazole, sulfapyridine, sulfamerazine, sulfamethoxydiazine, sulfamethylthiazole, sulfamethazine, sulfamonomethoxine, sulfamethoxypyridazine, sulfisoxazole, sulfamethoxazole, sulfadimethoxine and sulfaquinoxaline in natural animal casings by HPLC after solid-phase extraction. The sulfonamides were extracted with acetonitrile and the extract cleaned up with an Oasis MCX SPE cartridge prior to analysis. Separation was on a ZOBAX Eclipse XDB-C₈ column using gradient elution with acetonitrile/methanol/0.1% acetic acid. The effect of separation conditions on chromatographic behavior and recovery has been studied. Calibration graphs were linear with very good correlation coefficients (r = 0.9983−0.9996) in the concentration range from 0.02 to 1 μg mL⁻¹. The limits of quantitation (LOQ) for the 13 sulfonamides were in the range of 1.5–2.2 μg kg⁻¹. Decision limits (CCα) and detection capabilities (CCβ) were in the range of 105.2–111.0 and 113.0–120.2 μg kg⁻¹, respectively. The recovery for casings spiked with 1.5–100 μg kg⁻¹ ranged from 65.2 to 85.9%. The relative standard deviations (RSDs) of the sulfonamides for six measurements at 100 μg kg⁻¹ were from 2.2 to 7.7%. The applicability of the method to the analysis of salted swine casings, salted sheep casings and dry casing samples was demonstrated.     

Determination of Pyrethroid Insecticide Deltamethrin by Micellar Liquid Chromatography with Spectrophotometric Detection

A simple micellar liquid chromatographic technique for deltamethrin determination was developed and validated. The method provided to be suitable for deltamethrin determination in pediculicide shampoo. Kromasil C18 column (150 mm×4.6 mm, 5 μm) and mobile phase −0.12 M sodium dodecyl sulfate with 9% (v/v) 1-butanol were used for deltamethrin separation. Detection wavelength was 265 nm. The retention time was about 15 min. Different validation parameters were evaluated. The specificity of the method was demonstrated. Linearity was established in the range 10–40 μg L⁻¹. The limits of detection and quantitation were 1.06 and 3.22 μg mL⁻¹, respectively. The method showed excellent accuracy (100.6%) and precision (repeatability) gave a relative standard deviation of less than 1%. The influence of the various method parameters (robustness study) was also studied.     

Simultaneous HPLC Analysis of Olmesartan and Hydrochlorothiazide in Combined Tablets and in vitro Dissolution Studies

A simple, rapid and reproducible HPLC method was developed and validated for the simultaneous determination of olmesartan (OLM) medoxomil and hydrochlorothiazide (HCT) in combined tablets. Chromatography was carried out on a 4.6 mm I.D × 200 mm, 5 μm cyano column with methanol–10 mM phosphoric acid containing 0.1% triethylamine (pH 2.5, 50:50 v/v) at a flow rate of 1.0 mL min⁻¹ and UV detector was set at 260 nm. Valsartan (VAL) was used as internal standard (IS). A linear response was observed in the range of 0.2–6 μg mL⁻¹ (r ² = 0.9998) for OLM and 0.1–4 μg mL⁻¹ (r ² = 0.9999) for HCT, respectively. The method showed good recoveries (99.56% for OLM and 99.48% for HCT) and the relative standard deviation (RSD) values for intra- and inter-day precision were 0.70–1.59 and 0.80–2.00% for OLM and 1.20–1.37 and 1.63–1.93% for HCT, respectively. The developed method was applied successfully for quality control assay of OLM and HCT in combined tablets and in vitro dissolution studies.     

Stereospecific analysis of flurbiprofen and its major metabolites in plasma and urine by chiral-phase liquid chromatography

Summary Direct chiral-phase HPLC methods have been developed for the determination of flurbiprofen and its major metabolites, namely 4′-hydroxyflurbiprofen and 3′-hydroxy-4′-methoxyflurbiprofen, in biological fluids using a derivatized amylose chiral stationary phase (CSP; Chiral-pak AD). Quantification of all three analytes, both free and conjugated, in urine was carried out following liquid-liquid extraction using tandem ultraviolet (UV) and fluorescence detection. Determination of flurbiprofen and the 4′-hydroxy-metabolite in plasma utilized the same CSP but required modification in the mobile phase composition and sole use of fluorescence detection. The urine assay was linear (r>0.998) between 0.05–10 μg mL⁻¹, 0.1–20 μg mL⁻¹ and 0.01–2 μg mL⁻¹ for the enantiomers of flurbiprofen, 4′-hydroxyflurbiprofen and 3′-hydroxy-4′-methoxyflurbiprofen respectively. The plasma assay was linear (r>0.997) between 0.1–6 μg mL⁻¹ and 0.01–0.6 μg mL⁻¹ for the enantiomers of flurbiprofen and 4′-hydroxyflurbiprofen respectively. Both assays, typically yielded within- and between-day imprecision and accuracy values less than 10% for the enantiomers of the different analytes. Initial volunteer studies suggest that the disposition of flurbiprofen displays modest enantioselectivity in humans.     

Reversed Capillary Electrochromatography: Investigation of Peak Symmetry for Eluting Curves of Solutes

Summary A clean method without use of organic solvents has been developed for isolation and high-performance liquid chromatographic (HPLC) determination of oxytetracycline (OTC) and sulphadimidine (SDD) in cow's milk. Isolation is rapid and simple—homogenization with an inorganic acid solution by means of a handy ultrasonic homogenizer, which is easy-to-use and portable, followed by centrifugation. Reversed-phase HPLC was performed on a C₄ column, with 1.25 mmol L⁻¹ succinic acid solution as mobile phase, and identification was by means of a photodiode-array detector. Separation of the analytes was achieved in less than 8 min. Significant linearity was established over the concentration range of 0.1–1.0 μg mL⁻¹ for both target compounds (r>0.99,P<0.01). Average recoveries of OTC and SDD (each spiked at 0.1–1.0 μg mL⁻¹) were ≥88.8, and inter- and intra-assay variability was ≤2.8%. The total time required for analysis of one sample was <20 min. The limits of quantitation of the method (μg mL⁻¹ in milk) were 0.044 for OTC and 0.023 for SDD. No organic solvent was used at any stage of the analysis.     

Simple and rapid determination of piperacillin and ceftazidime in human plasma samples by HPLC

Summary A simple and rapid liquid chromatographic method has been developed for the determination of therapeutic levels of piperacillin (I) and ceftazidime (II) in human plasma. Plasma and p-propionamidophenol (internal standard) were precipitated with methanol (I) or 20% trichloroacetic acid (II). The supernatant was analysed on a 5 μm Spherisorb ODS C₁₈ column with acetonitrile-0.05 M phosphate buffer pH 3.8 as mobile phase and ultraviolet detection at 254 nm. The calibration graph was linear from 10 to 250 μg mL⁻¹, for (I), and from 5 to 200 μg mL⁻¹ for (II). Intra and inter-day CV did no exceed 2.29% for (I), and were 10.76–11.13%–2.00–5.62 for (II) at concentrations of 10 μg mL⁻¹ and 250 μg mL⁻¹.     

On-line determination of bipyridylium herbicides in water by HPLC

Summary Selective on-line solid phase extraction (SPE) and liquid chromatography determination (HPLC) of diquat, paraquat and difenzoquat from environmental water samples has been accomplished with Graphitized Carbon Black (GCB) as both extraction and analytical columns. The method involved passing of 50 mL of water through a cartridge filled with Carbograph. In the elution step, the herbicides were transferred from the cartridge to the analytical column (Hypercarb) by mean of a gradient of pH 3 aqueous solution of tetramethylammonium hydroxide (TMAOH) and ammonium sulphate and methanol. Hypercarb columns were found to give a low probability of false positives for bypiridylium herbicides and are very selective for polar compounds. Recovery was better than 80 %. The breakthrough volume was studied with distilled water spiked with the herbicides at various concentration levels (from 0.1 to 20 μg L⁻¹). The limits of quantification of the method were lower than 0.1 μg L⁻¹. The total analytical method was applied to surface waters from Torreblanca Nature Park (Castelló, Spain). Presented at the 21^st ISC held in Stuttgart, Germany, 15^th–20^th September, 1996.     

Simultaneous Determination of Olanzapine and Fluoxetine by HPLC

A rapid, specific reversed phase HPLC method has been developed for simultaneous determination of olanzapine and fluoxetine in their formulations. Chromatographic separation of these two pharmaceuticals was carried out on an Inertsil C₁₈ reversed phase column (150 mm × 4.6 mm, 5 μm) with a 40:30:30 (v/v/v) mixture of 9.5 mM sodium dihydrogen phosphate (pH adjusted to 6.8 ± 0.1 with triethylamine), acetonitrile and methanol as mobile phase. The flow rate 1.2 mL min⁻¹ and the analytes are monitored at 225 nm. Paroxetine was used as internal standard. The assay results were linear from 25 to 75 μg mL⁻¹ for olanzapine (r ² ≥ 0.995) and 100–300 μg mL⁻¹ for fluoxetine (r ² ≥ 0.995), showed intra- and inter-day precision less than 1.0%, and accuracy of 97.7–99.1% and 97.9–99.0%. LOQ was 0.005 and 0.001 μg mL⁻¹ for olanzapine and fluoxetine, respectively. Separation was complete in less than 10 min. Validation of the method showed it to be robust, precise, accurate and linear over the range of analysis.     

Determination of biogenic amines in food by automated pre-column derivatization with 2-naphthyloxycarbonyl chloride (NOC-CI)

Summary The fluorogenic reagent 2-naphthyloxycarbonyl chloride (NOC-Cl) has been used for the automated precolumn derivatization of biogenic amines (BAs) at ambient followed by liquid-chromatographic separation of the derivatives formed. For optimized derivatization samples in 0.5 M borate buffer (pH 9.0) were derivatized with 5 mM NOC-Cl in acetonitrile (MeCN) for 3 minutes. Excess of reagent was scavenged by addition of 20 mM glycine in water. For HPLC a Superspher^? RP-18e column and gradient elution using 0.1 M sodium acetate buffer (pH 4.4) and MeCN were used. The NOC-derivatives were detected by fluorescence at an emission wavelength of 335 nm at an excitation wavelength of 274 nm. This method allows the detection of BAs (2-phenylethylamine, putrescine, histamine, cadaverine, tyramine, spermidine, spermine) found in food and beverages (fruit juices, wines, various vinegars, fermented cabbage juice, and salmon). Detection limit of BAs are approximately 49–113 μg kg⁻¹ with the exception of histamine (747 μg kg⁻¹) (injected amounts: 18–41 pg histamine 267 pg), at a signalto-noise ratio of 3:1. The limits of determination are approximately 82–189 μg kg⁻¹ (histamine 1245 μg kg⁻¹) at a signal-to-noise ratio of 5:1. The correlation coefficients of linearity are 0.9910–0.9976. Recoveries from different matrices range from 65 to 109%, depending on the sample investigated. Presented at the 21^st ISC held in Stuttgart, Germany, 15^th–20^th September, 1996.     

Determination of Trihalomethanes in Drinking Water by Dispersive Liquid–Liquid Microextraction then Gas Chromatography with Electron-Capture Detection

Dispersive liquid–liquid microextraction (DLLME) has been used for preconcentration of trihalomethanes (THMs) in drinking water. In DLLME an appropriate mixture of an extraction solvent (20.0 μL carbon disulfide) and a disperser solvent (0.50 mL acetone) was used to form a cloudy solution from a 5.00-mL aqueous sample containing the analytes. After phase separation by centrifugation the enriched analytes in the settled phase (6.5 ± 0.3 μL) were determined by gas chromatography with electron-capture detection (GC–ECD). Different experimental conditions, for example type and volume of extraction solvent, type and volume of disperser solvent, extraction time, and use of salt, were investigated. After optimization of the conditions the enrichment factor ranged from 116 to 355 and the limit of detection from 0.005 to 0.040 μg L⁻¹. The linear range was 0.01–50 μg L⁻¹ (more than three orders of magnitude). Relative standard deviations (RSDs) for 2.00 μg L⁻¹ THMs in water, with internal standard, were in the range 1.3–5.9% (n = 5); without internal standard they were in the range 3.7–8.6% (n = 5). The method was successfully used for extraction and determination of THMs in drinking water. The results showed that total concentrations of THMs in drinking water from two areas of Tehran, Iran, were approximately 10.9 and 14.1 μg L⁻¹. Relative recoveries from samples of drinking water spiked at levels of 2.00 and 5.00 μg L⁻¹ were 95.0–107.8 and 92.2–100.9%, respectively. Comparison of this method with other methods indicates DLLME is a very simple and rapid (less than 2 min) method which requires a small volume of sample (5 mL).