A fully automated amino acid analyzer using NBD-F as a fluorescent derivatization reagent

A fully automated amino acid analyzer using NBD-F (4- fluoro-7-nitro-2,1,3-benzoxadiazole) as a fluorescent derivatization reagent was developed. The whole analytical process was fully automated from derivatization, injection to HPLC separation and quantitation. The derivatization reaction conditions were re-evaluated and optimized. Amino acids were derivatized by NBD-F for 40 min at room temperature in the borate buffer (pH 9.5). The derivatives were separated within 100 min and fluorometrically detected at 540 nm with excitation at 470 nm. The detection limits for amino acids were in the range of 2.8-20 fmol. The calibration curves were linear over the range of 20 fmol to 20 pmol on column with the correlation coefficients of 0.999. The coefficients of variation were less than 5% at 3 pmol injection for all amino acids. Amino acids in rat plasma were determined by the proposed HPLC method.     

Ultramicro Analysis of Reducing and Non-Reducing Sugars by Liquid Chromatography

Abstract

A post-column detection system for ultramicro amount of sugars has been developed using taurocyamine as the labelling reagent. Less than 10 pmol of reducing sugars were determined by this HPLC system. Non-reducing sugars were detected by the addition of periodate to the reagent.

Modification of the reaction reagent components made this detection system feasible to apply to various methods for separation of carbohydrates using pure water, acetonitrile/water mixtures, borate buffer or aqueous sodium hydroxide as the eluent.     

Catecholamines derivatized with 4-fluoro-7-nitro-2,1,3-benzoxadiazole: characterization of chemical structure and fluorescence properties

4-Fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F) was evaluated as a fluorogenic derivatization reagent for the analysis of the catecholamines, dopamine, epinephrine, norepinephrine, and their naturally occurring metabolites, metanephrine and normetanephrine, homovanillic acid, 3,4-dihydroxyphenyl acetic acid. These compounds reacted rapidly with NBD-F under mild conditions to form stable derivatives. The optimal reaction conditions were found to be 12.5 mM borate buffer pH 8.0 in water:acetonitrile (1:1) at 50 °C for 5 min. New NBD derivatives of all the catecholamines and metabolites were prepared and purified and were shown by electrospray mass spectrometry to be fully reacted at all available catechol and amine sites, resulting in di- or tri-substituted derivatives. Homovanillic acid and 3,4-dihydroxyphenyl acetic acid reacted with NBD-F but gave non-fluorescent derivatives. The fluorescence excitation wavelength maximum demonstrated a red shift for the derivatives with increasing polarity of the solvent and the fluorescence intensity increased linearly with increasing organic ratio in the solvent-aqueous buffer complex. The presence of electrolyte in the solvent and the electrolyte concentration in the solvent-electrolyte complex had little effect on the fluorescent intensity. The fluorescence quantum yields in acetonitrile were also obtained. The separation behavior of the NBD-catecholamines was determined by high-performance liquid chromatography (HPLC). The studies demonstrated good potential for the application of NBD-F derivatization to the quantitative analysis of catecholamines and related compounds in biological matrices.     

Separation of Ribose-5-phosphate,Ribose-1-phosphate,Deoxyribose-1-phosphate by High Performance Liquid Chromatography and Spectrophotometric Determination Using 2-Cyanoacetamide

Abstract

A simple procedure for separation of ribose-5-phosphate, deoxyribose-1-phosphate and ribose-1-phosphate is based on high performance liquid chromatography using reversed phase 4 × 300 mm “μ Bondapak/NH₂” column. The column is equilibrated with 0.13 M borate buffer (pH 7.5) followed by gradient elution of ribose-5-phosphate, deoxyribose-1-phosphate and ribose-1-phosphate using water, 0.05 M borate buffer containing 0.1 M MgCl₂ (pH 9.6) and 0.05 M sodium acetate-acetic acid buffer containing 0.1 M MgCl₂ (pH 5.0) as eluants respectively. Eluates of borate complex “μ Bondapak/NH₂” column are brought to pH 9.6 by the addition of 1 N KOH and enzymatically hydrolysed with alkaline phosphatase (EC 3.1.3.1) to release the free pentoses. The free pentoses are mixed with a reagent solution prepared from aqueous solution of 2% cyanoacetamide and 0.6 M borate buffer (pH 9.6), and the mixture is boiled for 10 minutes and the absorbance of the product is measured at 276 nm using a spectrophotometer.     

Determination of carbohydrates as their 3-aminophthalhydrazide derivatives by capillary zone electrophoresis with on-line chemiluminescence detection

A method based on pre-capillary derivatization with luminol (3-aminophthalhydrazide) for carbohydrate analysis using capillary electrophoresis with on-line chemiluminescence (CL) detection was developed. The derivatives of seven monosaccharides were separated and detected by using 200 mM borate buffer containing 100 mM hydrogen peroxide at pH 10.0 as separation electrolyte and 25 mM hexacyanoferrate in 3 M sodium hydroxide solution as post-capillary chemiluminescence reagent with separation efficiencies ranging from 160,000 to 231,000 plates per metre. The minimum amount of carbohydrate derivatized was 2 pmol (corresponding to the concentration of 2 microM). The method also provided a linear response for glucose in the concentration range of 0.1-250 microM with a mass detection limit of 420 amol or a concentration detection limit of 0.1 microM. Preliminary work using the CE-CL format to determine glucose in a rat brain microdialysis sample is presented as a typical case.     

Simultaneous analysis of sulfur-containing excitatory amino acids using micellar electrokinetic chromatography with diode array and laser-induced fluorescence detection

The suitability of micellar electrokinetic chromatography (MEKC) coupled with diode array or laser-induced fluorescence (LIF) detection to analyze the four sulfur-containing excitatory amino acids (SEAA), homocysteine sulfinic acid (HCSA), homocysteic acid (HCA), cysteine sulfinic acid (CSA), and cysteic acid (CA) was investigated. 5-Carboxy-fluorescein succinimidyl ester was chosen as fluorescent reagent to derivatize HCSA, HCA, CSA, and CA. During method development, the yield of reaction dependent on pH and incubation time as well as the stability of the products were analyzed. The maximum yield was obtained after 30 min using a 0.1 M borate buffer (pH 8.9) as derivatization buffer. Each labeled amino acid exhibited high stability at room temperature over a period of 5 days. Baseline separation of labeled HCSA, HCA, CSA, and CA was obtained using a buffer consisting of 0.1 M borate, 50 mM sodium dodecyl sulfate (SDS), and 5% v/v methanol (pH 9.0). By applying LIF detection, limits of detection ranged from 0.9 x 10(-10) M for HCSA to 6.0 x 10(-10) M for CA, respectively. Slightly modified separation conditions enabled the analysis of SEAA in cerebrospinal fluid in the presence of the neurotransmitters glutamate and aspartate. In conclusion, MEKC coupled with LIF detection is a suitable technique for the simultaneous and sensitive analysis of SEAA. Further work will focus on the validation of the method with cerebrospinal fluid as sample matrix.     

毛细管电色谱-激光诱导荧光检测法分析食品中的生物胺

以4-氟-7-硝基-2,1,3-苯并氧杂恶二唑(NBD-F)为衍生化试剂,建立了食品中5种痕量生物胺(色胺、组胺、酪胺、亚精胺、精胺)的毛细管电色谱-激光诱导荧光检测(CEC-LIF)分析方法。采用50 mmol/L硼酸盐缓冲溶液(pH 8.0)作为衍生介质,在75℃条件下对生物胺进行衍生化反应25 min。生物胺衍生产物的最优色谱条件:固定相为C18毛细管电色谱柱,流动相为乙腈-乙酸铵(20 mmol/L,pH 8.0)(75∶25,v/v),辅助压力为6.9 MPa,分离电压为-8 kV,流速为0.03 mL/min。实验结果表明,生物胺的检出限(LOD,S/N=3)为0.1~1.0μg/L,加标回收率为78.3%~113.9%。该方法可成功用于加工和发酵食品中生物胺的测定,结果与传统HPLC法的检测结果无显著性差异,且检出限更低、分析速度更快,对于食品中痕量污染物的残留监测具有应用价值。     

Simultaneous liquid chromatographic assay of amantadine and its four related compounds in phosphate-buffered saline using 4-fluoro-7-nitro-2,1,3-benzoxadiazole as a fluorescent derivatization reagent

Simultaneous HPLC assay of 1-adamantanamine hydrochloride (amantadine) and its four related compounds [2-adamantanamine hydrochloride (2-ADA), 1-adamantanmethylamine (ADAMA), 1-(1-adamantyl)ethylamine hydrochloride (rimantadine) and 3,5-dimethyl-1-adamantanamine hydrochloride (memantine)] in phosphate-buffered saline (pH 7.4) after pre-column derivatization with 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F) was developed. Phosphate-buffered saline samples were mixed with borate buffer and NBD-F solution in acetonitrile at 60 degrees C for 5 min and injected into HPLC. Five derivatives were well separated from each other. The lower limits of detection of amantadine, 2-ADA, ADAMA, rimantadine and memantine were 0.008, 0.001, 0.0008, 0.0015 and 0.01 microg/mL, respectively. The coefficients of variation for intra- and inter-day assay were less than 6.4 and 8.2%, respectively. The method presented was applied to a binding study of these compounds to human alpha(1)-acid glycoprotein. While affinity constants and capacities for ADAMA, rimantadine and memantine were calculated by means of Scatchard plots, those for the others were not determined. ADAMA, rimantadine and memantine were bound with different affinities and capacities. These results indicate that NBD-F is a good candidate as a fluorescent reagent to simultaneously determine amantadine and its four related compounds by HPLC after pre-column derivatization. Our method can be applied to binding studies for protein.     

Fluorescent detection of peptides and amino acids for capillary electrophoresis via on-line derivatization with 4-fluoro-7-nitro-2,1,3-benzoxadiazole

An in-capillary derivatization of amino acids and peptides with 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F) was developed for their subsequent capillary electrophoretic analysis with laser-induced fluorescence detection (λ _ex=488 nm). The in-capillary derivatization was achieved in zone-passing mode by introducing successive plugs of sample and NBD-F into a fused silica capillary previously equilibrated with an alkaline borate buffer. To prevent NBD-F hydrolysis and to achieve a reliable derivatization, NBD-F was prepared daily in absolute ethanol and a plug of absolute ethanol was introduced between the sample and NBD-F reagent plugs. Various parameters influencing the derivatization efficiency were investigated and the optimum conditions were as follows: background electrolyte (BGE), 20 mM borate buffer (pH 8.8); introduction time, 4 s for sample and 2 s for NBD-F; molar ratio of NBD-F/sample, above 215; temperature, 45 °C for amino acids and 35 °C for peptides; applied voltage, +15 kV. The validation of the in-capillary derivatization method under optimal conditions showed a good linearity between the heights of the derivative peaks and the concentrations of the amino acids. The intra-day relative standard deviations of the migration times and the peak heights were less than 1.3% and 4.6%, respectively. The efficient derivatization and separation of a mixture of valine, alanine, glutamic acid and aspartic acid were achieved using this technique. Peptides such as buccaline and β-protein fragment 1–42 could also be derivatized using the developed in-capillary derivatization procedure. In‑capillary derivatization and separation of amino acids with different concentrations. From the top to bottom the concentrations are 1.11×10⁻⁵ M, 5.55×10⁻⁶ M, 2.78×10⁻⁶ M, 6.95×10⁻⁷ M. for valine; 1.26×10⁻⁵ M, 6.30×10⁻⁶ M, 3.15×10⁻⁶ M, 7.88×10⁻⁷ M for alanine; 3.78×10⁻⁵ M, 1.89×10⁻⁵ M, 9.45×10⁻⁶ M, 2.36×10⁻⁶ M for glutamic acid;, 4.27×10⁻⁵ M, 2.14×10⁻⁵ M, 1.07×10⁻⁵ M, 2.68×10⁻⁶ M for aspartic acid. Experiment conditions: injection order: 4s for sample, 1s for absolute ethanol, and then 2s for 5.24×10⁻² M NBD‑F; BGE: 20 mM borate pH 8.77; Applied voltage: 15 kV.     

Amino acid analysis by micellar electrokinetic chromatography with laser-induced fluorescence detection: application to nanolitre-volume biological samples from Arabidopsis thaliana and Myzus persicae

Amino acids were derivatised with 4-fluoro-7-nitrobenzo-2,1,3-oxadiazol (NBD-F), separated by micellar electrokinetic chromatography (MEKC), and detected by argon-ion (488 nm) laser-induced fluorescence. The optimised MEKC background electrolyte conditions were: 40 mM sodium cholate, 5 mM beta-cyclodextrin in 20 mM aqueous borate buffer, pH 9.1, with 7% v/v acetonitrile. Using these conditions, 19 amino acids were separated within 17 min. The limits of detection were in the range of 7.6-42.2 pmol/mL and limits of quantitation from 0.05-0.14 nmol/mL. The method was systematically validated for injection volume error, migration time variation, calibration linearity, accuracy, precision, and recovery. Nanolitre volume samples of phloem sap of individual sieve element cells from the plant Arabidopsis thaliana and honeydew from the aphid Myzus persicae were directly analysed with this method. Quantitative amino acid concentrations in these two biological matrices were profiled for the first time. This method is particularly important because it allows the complete profile of the amino acids obtained from individual phloem elements, allowing cell to cell and plant to plant variation to be quantified, which to date has not been possible with Arabidopsis thaliana.     

Effect of borate buffer on the photolysis of riboflavin in aqueous solution

The photolysis of riboflavin (RF) in the presence of borate buffer (0.1-0.5M) at pH 8.0-10.5 has been studied using a specific multicomponent spectrophotometric method for the determination of RF and photoproducts, formylmethylflavin (FMF), lumichrome (LC) and lumiflavin (LF). The overall first-order rate constants for the photolysis of RF (1.55-4.36 x 10(-2)min(-1)) and the rate constants for the formation of FMF (1.16-3.52 x 10(-2)min(-1)) and LC (0.24-0.84 x 10(-2)min(-1)) have been determined. The values of all these rate constants decrease with an increase in buffer concentration suggesting the inhibition of photolysis reaction by borate species. The kinetic data support the formation of a RF-borate complex involving the ribityl side chain to cause the inhibition of photolysis. The second-order rate constants for the borate inhibited reaction range from 1.17-3.94 x 10(-2)M(-1)min(-1). The log k-pH profiles for the reaction at various buffer concentrations indicate a gradual increase in rate, with pH, up to 10 followed by a decrease in rate at pH 10.5 probably due to ionization of RF and quenching of fluorescence by borate species. A graph of second-order rate constants against pH is a sigmoid curve showing that the rate of photolysis increases with an increase in pH. The results suggest the involvement of excited singlet state, in addition to excited triplet state, in the formation of LC.     

Microdetermination of hyaluronic acid in human urine by high performance liquid chromatography.

A high performance liquid chromatographic (HPLC) system is described for determination of the unsaturated disaccharide (delta Di-HA) derived from hyaluronic acid (HA) in human urine by digestion with hyaluronidase SD. The effects of eluents on the separation of delta Di-HA and delta Di-0S, which is derived from the reaction of chondroitin with the enzyme, have been studied. The established chromatographic conditions were as follows--column: a stainless steel tube (4 mm i.d. x 250 mm) packed with TSKgel NH2-60; eluent: a mixture of acetonitrile and 0.1 M Tris-HCl buffer containing 0.1 M boric acid and 10 mM sodium sulphate, pH 7.0 (64:36, v/v). The strong fluorescence of unsaturated disaccharide after the reaction with 2-cyanoacetamide in alkaline medium was used for post-column detection. The calibration curve for delta Di-HA was linear in the range 5 pmol-5nmol with a practical detection limit of 2 pmol. The assay coefficients of variation (n = 5) at 200 pmol for delta Di-HA and delta Di-0S were 1.7 and 1.5%, respectively. This HPLC system has been applied to the determination of HA in human urine.     

High-performance liquid chromatographic assay for pteroylpolyglutamate hydrolase

A highly sensitive assay for pteroylpolyglutamate hydrolase is described employing high-performance liquid chromatography (HPLC) with ultraviolet detection at 280 nm. The method is based on the separation of pteroylpolyglutamates containing various glutamyl residues on a C18 muBondapak reversed-phase column. Individual pteroylpolyglutamates are eluted by a gradient of 2.5-8.5% acetonitrile in 0.1 M potassium phosphate buffer (pH 6.0) within 20 min. The polyglutamates with higher glutamyl residues were less well retained in the reversed-phase column. The relationship between the peak area and the amount of pteroylpolyglutamate was observed to be linear over the range 10 pmol to 2.5 nmol. Human serum pteroylpolyglutamate hydrolase was studied using pteroylpentaglutamate as substrate in 0.1 M sodium acetate buffer (pH 4.5). The enzyme appeared to function as an exopeptidase based on the detection of intermediates, pteroyltetra-, tri-, and -diglutamate, and the product, pteroylmonoglutamate. Using the HPLC assay, extracts of Plasmodium falciparum were found not to contain detectable enzyme activity.     

Chromatographic determinations of a beta-lactam antibiotic, cefaclor by means of fluorescence, chemiluminescence and mass spectrometry

We report analytical informations on the quantification of cefaclor (CCL), a beta-lactam antibiotic by three detection methods. The methods were based on the chemical derivatization of the drug with 4-(2'-cyanoisoindolyl)phenylisothiocyanate (CIPIC) under the reaction conditions with heating at 80 degrees C for 7 min in the presence of pyridine. The CIPIC reagent could react with the primary amino group of the drug to form CIPIC-conjugated CCL. The derivative emitted not only fluorescence (FL) at maximum emission wavelength of 410 nm with the irradiation at 310 nm excitation, but also chemiluminescence (CL) in the presence of H(2)O(2), borate buffer (pH 9.5) and acetonitrile. After reversed-phase liquid chromatographic separation of the CIPIC-conjugated CCL in blood, the derivative could be monitored with a FL detector, indicating the detection limit (S/N=3) of 10 pmol/injection. The CIPIC-conjugated CCL was further monitored most sensitively by a CL detector after simply mixing H(2)O(2) and borate buffer with the column eluate. The CL detection limit was 1.0 pmol/injection. In addition, we attempted to detect the CIPIC-conjugated CCL by liquid chromatographic-mass spectrometry (LC/MS). The MS method permitted the specific detection of the CIPIC derivative of the drug, though the sensitivity was 10(4)-times lower than that of the CL detection.     

Optimizing high-performance liquid chromatography method for quantification of glucosamine using 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate derivatization in rat plasma: application to a pharmacokinetic study

A sensitive and reliable HPLC method with fluorescence detection based on the precolumn derivatization of glucosamine with 6-aminoquinolyl-N-hydroxylsuccinimidyl carbamate (AQC) was established for the quantitative determination of glucosamine in rat plasma. The plasma protein was precipitated by acetonitrile, followed by vortex mixing and centrifugation. The supernatant was divided into the organic layer and aqueous layer by adding sodium chloride, and then the aqueous layer was derivatized with AQC in 0.2 M borate buffer of pH 8.8 before the HPLC analysis. An amino acid analysis column (3.9 x 150 mm, 4 microm) was applied, with 140 mM sodium acetate buffer (pH = 5.25) and acetonitrile as mobile phase at a flow rate of 1 mL/min. A linear correlation coefficient of 0.9987 was calculated within the range of 0.1-30 microg/mL of the standard curve for glucosamine. The limit of detection was 30 ng/mL. The intra- and inter-day precisions (as RSD) were less than 7.38 and 12.72%, respectively. The intra- and inter-day accuracy ranged from 91.8 to 110.0%. Extraction recoveries of glucosamine in plasma were more than 90%. The validated method was successfully applied for the quantitative determination of glucosamine in rat plasma and evaluation for pharmacokinetic study of glucosamine. It was also possible to be applied for the quantitative determination of other compounds containing amino group in biological samples.     

Separation and determination of epinephrine and dopamine in traditional Chinese medicines by micellar electrokinetic capillary chromatography with laser induced fluorescence detection

A micellar electrokinetic capillary chromatography method with laser-induced fluorescence detection was developed for the analysis of epinephrine and dopamine after derivatization with 4-chloro-7-nitrobenzo-2-oxa-1,3-diazole. The optimum derivatization conditions were: 30 mM sodium borate (pH adjusted to 8.0 with 1.0 M HCl), reaction time 30 min at 60 degrees C. Baseline separation was achieved within 14 min with a running buffer composed of 10 mM sodium borate + 25 mM sodium dodecyl sulfate (pH adjusted to 9.5 with 0.1 M NaOH) and an applied voltage of 15 kV. Good linearity relationships (correlation coefficients: 0.9991 for epinephrine and 0.9985 for dopamine) between peak areas and concentrations of the analytes were obtained. The detection limits and quantification limits for epinephrine and dopamine were 0.0038 mg/L and 0.013 mg/L, and 0.065 mg/L and 0.020 mg/L, respectively. The method was applied to the analysis of the two compounds in two Chinese medicines with recoveries in the range of 92.6-108.7%.     

毛细管电色谱-激发诱导荧光检测动物性食品中多肽类抗生素

以4-氟-7-硝基-2,1,3-苯并呋咱（NBD-F）为荧光试剂,建立了毛细管电色谱-激光诱导荧光（CEC-LIF）检测动物性食品中痕量杆菌肽、多粘菌素B和粘杆菌素等环状多肽抗生素的分析方法。在50 mmol/L的硼酸缓冲液（pH 7.5）中,多肽类抗生素经60℃衍生反应45 min。所得衍生产物采用苯基毛细管色谱填充柱进行分离,流动相为乙腈-磷酸盐缓冲液（pH 5.0,10 mmol/L）（55∶45,v/v）,辅助压力为3.8 MPa,分离电压为-10 kV,流速为0.02 mL/min。结果表明,多肽类抗生素的检出限（S/N=3）为5.0~10.0 ng/mL,满足目标物最大残留限量的检测要求。方法应用于饲料和牛奶样品的分析,平均回收率为72.9%~112.4%。该方法预处理操作简单、灵敏,为动物性食品中兽药残留分离分析提供了新手段。     

毛细管胶束电动色谱法分析PTH氨基酸

建立了用毛细管胶束电动色谱法（MEKC）分离19种PTH氨基酸的方法,并探讨了电压、pH值、温度、胶束浓度对氨基酸迁移时间的影响。方法具有速度快、灵敏度高、样品用量少的优点。     

Fluorimetric determination of kynurenine in human serum by high-performance liquid chromatography coupled with post-column photochemical reaction with hydrogen peroxide

A high-performance liquid chromatographic method involving post-column photochemical reaction and fluorimetric detection has been developed for the determination of kynurenine in serum. Kynurenine was separated on a column of Capcell Pak C18 (resistant to pH 10). The mobile phase consisted of 0.05 M Na2B4O7-0.1 M KH2PO4 buffer (pH 8.5)-ethanol (97:3, v/v) containing 60 mM hydrogen peroxide. The post-column reagent, containing 60% (v/v) ethanol, was mixed with the mobile phase, which was irradiated with ultraviolet light to induce fluorescence. The recovery of kynurenine was 95.9 +/- 5.0% (n = 6). The method allows the determination of as little as 2 pmol of kynurenine.     

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.