Pre-column Derivatization High-performance Liquid Research Articles

Determination of Steady-State Kinetic Parameters of 1-Deoxy-<I>D</I>-xylulose-5-phosphate Synthase by Pre-column Derivatization High Performance Liquid Chromatography Using 2,4 Dinitrophenylhydrazine as Derivative Reagent

利用含羰基化合物与2,4-二硝基苯肼在酸性条件下反应得到的产物腙对紫外-可见光有吸收的特性,采用柱前衍生高效液相色谱法测定了1-脱氧-D-木酮糖-5-磷酸合酶稳态动力学参数。酶反应产物1-脱氧-D-木酮糖-5-磷酸在碱性磷酸酶的作用下生成去磷酸化产物1-脱氧-D-木酮糖,然后与2,4-二硝基苯肼衍生成腙。衍生化反应的适宜条件为：HClO4浓度为1.5%,反应温度37℃,反应时间60 min,2,4-二硝基苯肼与1-脱氧-D-木酮糖-5-磷酸的摩尔比为6∶1。色谱条件：0～17 min,40%～80%甲醇;18～20 min,40%甲醇。结果表明,本方法具有较高的灵敏度和良好的线性关系,1-脱氧-D-木酮糖-5-磷酸的检出限为1.0 mg/L,在0.005～1.0 g/L范围内线性相关系数为0.999,相对标准偏差小于5.0%（n=3）,标准回收率为102.22%。用本方法测得的1-脱氧-D-木酮糖-5-磷酸合酶稳态动力学参数Km、Vmax和Kcat与文献报道一致。

Determination of Steady-State Kinetic Parameters of 1-Deoxy-D-xylulose-5-phosphate Synthase by Pre-column Derivatization High Performance Liquid Chromatography Using 2,4-Dinitrophenylhydrazine as Derivative Reagent

Abstract Carbonyl containing compounds can normally be determined by high performance liquid chromatography (HPLC) through pre-column derivatization with 2,4-dinitrophenylhydrazine (DNPH). Using this method, the steady-state kinetic parameters of 1-deoxy- D -xylulose-5-phosphate synthase (DXS) was measured. First, the enzymatic product 1-deoxy- D -xylulose-5-phosphate (DXP) was dephosphorylated by alkaline phosphatase, then the product 1-deoxy- D -xylulose (DX) was derived with DNPH in acidic solution to give the corresponding hydrazones which was subsequently determined by HPLC. The optimum derivatization conditions are as follows: 1.5% perchloric acid, reaction temperature of 37 °C, reaction time of 60min, molar ratio of DNPH to DXP 6:1. The HPLC was run with a linear gradient of methanol-water solvent system: 0 min, 40% methanol; 17 min, 80% methanol; 18 min, 40% methanol; 20 min, 40% methanol. The method has a detect limit of 1 mg L −1 for DXP and a linear correlation in the range of 0.005–1 g L −1 ( R = 0.999). The relative standard deviation is less than 5.0%. The steady-state kinetic parameters of DXS determined by this method are identical with the reported data.

Online Pre-Column Derivatization with Chromatographic Separation to Determine Folic Acid

A simple, sensitive, and selective online pre-column derivatization high-performance liquid chromatographic method was developed and validated for the first time to determine trace levels of folic acid (FA). An oxidant cerium (IV) trihydroxyhydroperoxide packed reactor was used for pre-column oxidation and was combined by column switching with a C18 analytical column for sample enrichment and separation. The method was based on oxidative cleavage of FA into highly fluorescence products, 2-amino-4-hydroxypteridine-6-carboxaldehyde and the corresponding 2-amino-4-hydroxypteridine-6-carboxylic acid, during the flow of 0.04 M phosphate buffer (pH 3.5) containing the analyte through packed reactor at a flow rate of 0.2 mL/min and 40°C. The fluorescent products were enriched on the head of the analytical column for the final separation. The separation was performed at room temperature using a mobile phase consisting of phosphate buffer (0.04 M, pH 3.5) and acetonitrile (90:10, v/v). The eluents were monitored at emission and excitation wavelengths of 463 and 367 nm, respectively. The method showed excellent recovery, precision and accuracy with detection limits of 0.067 ng/mL from 500 µL of sample FA. The developed method was successfully applied to the determination of FA in pharmaceutical formulations and showed a recovery of 99.31% and a relative standard deviation of 1.72%.

Determination of naftopidil enantiomers in rat plasma using chiral solid phases and pre-column derivatization high-performance liquid chromatography

Two bioanalytical HPLC methods (chiral solid phases (CSPs) HPLC and pre-column derivatization HPLC) were developed and validated for the determination of naftopidil enantiomers in rat plasma. Analytes were extracted from biomaterials by liquid–liquid extraction. The pre-column derivatization HPLC method employed (+)-diacetyl-l-tartaric anhydride (DATAN) as the pre-column derivatization reagent, and subsequent separation of diastereomers was conducted on an Agilent Hypersil ODS column with a mixture of methanol–acetonitrile–phosphate buffer (pH 4.1; 20mM) (40:30:30, v/v/v) flowing at 1mL/min as the mobile phase. The CSPs HPLC method utilized a Chiralpak IA column with a mobile phase of methanol–acetonitrile–acetate buffer (pH 5.3; 5mM) (50:25:25, v/v/v) flowing at 0.5mL/min. In both methods, the analytes were monitored using a fluorescence detector with an excitation wavelength of 290nm and an emission wavelength of 340nm. Both methods were consistent (RSD<15% by the derivatization method and<10% by the CSPs method) and linear (r>9950). Compared to the pre-column derivatization method, the CSPs method had lower quantification limits (10.6/9.6ng/mL of (+)-/(−)-naftopidil by derivatization method and 1.1/1.8ng/mL of (+)-/(−)-naftopidil by CSPs method), and was simpler to carry out. The validated CSPs method was successfully applied in a pharmacokinetic study of naftopidil enantiomers in rats, which showed that pharmacokinetic parameters of (+)- and (−)-NAF after intravenous administration of (±)-NAF were similar.

Formation of Reactive Impurities in Aqueous and Neat Polyethylene Glycol 400 and Effects of Antioxidants and Oxidation Inducers

A 2,4-dinitrophenylhydrazine (DNPH) precolumn derivatization high-performance liquid chromatography-ultraviolet detection (HPLC-UV) method was developed to quantify levels of formaldehyde and acetaldehyde in polyethylene glycol (PEG) solutions. Formic acid and acetic acid were quantified by HPLC-UV. Samples of neat and aqueous PEG 400 solutions were monitored at 40°C and 50°C to determine effects of excipient source, water content, pH, and trace levels of hydrogen peroxide or iron metal on the formation of reactive impurities. The effects of antioxidants were also evaluated. Formic acid was the major degradation product in nearly all cases. The presence of water increased the rate of formation of all impurities, especially formic acid as did the presence of hydrogen peroxide and trace metals. Acidic pH increased the formation of acetaldehyde and acetic acid. A distribution of unidentified degradation products formed in neat PEG 400 disappeared upon addition of HCl with corresponding increase of formic acid, indicating they were likely to be PEG-formyl esters. Other unidentified degradation products reacted with DNPH to form a distribution of derivatized products likely to be PEG aldehydes. Antioxidants butylated hydroxyanisole, butylated hydroxytoluene, propyl gallate d-alpha tocopheryl polyethylene glycol-1000 succinate, and sodium metabisulfite were effective in limiting reactive impurity formation, whereas ascorbic acid and acetic acid were not.

Determination of homocysteine in plasma by precolumn derivatization-high performance liquid chromatography with fluorescence detection

A precolumn derivatization-high performance liquid chromatographic method for the determination of homocysteine (Hcy) in plasma was established. Tris (2-carboxyethyl) phosphine hydrochloride (TCEP) and N-(1-pyrenyl) maleimide (NPM) were used as the reduced reagent and derivatization reagent, respectively. The separation was carried out on an Agilent Hypersil C-18 column (250 mm x 4.0 mm, 5 microm) in gradient elution mode. The mobile phase consisted of A (15 mmol/L sodium acetate solution), B (acetonitrile) and C (300 mL water containing 1 mL acetic acid and 1 mL phosphoric acid). The eluate was monitored by the fluorescence detector at an excitation wavelength of 330 nm and an emission wavelength of 380 nm. The mean recovery of Hcy was (102.08 +/- 4.94)%. The linear range was from 0.500 micromol/L to 100 micromol/L, with a detection limit of 0.016 micromol/L. The intra-day and inter-day relative standard deviations (RSDs) for Hcy were less than 5%. Seven plasma samples of patients with hypertension and seven plasma samples of healthy controls were tested, and the results demonstrated that the Hcy in the plasma from the hypertension group was significantly different from that of the control group (p < 0.05). The developed method is simple, fast, accurate, and suitable for clinical measurement.

Determination of Ciprofloxacin in Human Plasma and Urine by Precolumn Derivatization High Performance Liquid Chromatography with Fluorescence Detection

A sensitive, simple and selective high-performance liquid chromatographic (HPLC) method was developed for the determination of ciprofloxacin in biological fluids. The method is based on the reaction between the drug and 4-chloro-7-nitrobenzofurazan (NBD-Cl) in borate buffer of pH 9.0 to yield a highly fluorescent derivative that is measured at 535 nm after excitation at 464 nm. The calibration curves were linear over the concentration ranges of 25–3000 and 50–3000 ng·mL−1 for plasma and urine, respectively. The mean recovery of ciprofloxacin from plasma and urine was 98.37% and 98.40%, respectively. The method was found to be sensitive, precise, accurate, and reproducible. All of the validation parameters were within the acceptance range.

Comparasion of polysaccharides in parent root, daughter root and rootlet of Aconitum carmichaeli

To study the polysaccharides contents and monosaccharide compositions in parent root, daughter root and rootlet of Aconitum carmichaeli. The conversion coefficient of A. carmichaeli polysaccharide to glucose was obtained by refined polysaccharides, and then the contents of crude polysaccharides in parent root, daughter root and rootlet were determined by sulfuric-phenol spectrometry method; analysis of monosaccharide compositions in polysaccharides from A. carmichaeli was carried out by pre-column derivatization high performance liquid chromatography with 1-phenyl-3-methyl-5-pyrazolone (PMP). The contents of polysaccharides in parent root, daughter root and rootlet were 22.02%, 33.53% and 6.10%, respectively. Parent root, daughter root and rootlet mainly contained glucose, and in addition they contained a small amount of galacturonic acid, galactose and arabinose. Daughter root contained mannose yet, and rootlet still contained mannose, rhamnose and xylose. The method is simple, rapid, and accurate. The content of polysaccharide in rootlet is lowest, and monosaccharide compositions in rootlet are significantly different from parent root and daughter root.

Highly sensitive and selective pre-column derivatization high-performance liquid chromatography approach for rapid determination of triterpenes oleanolic and ursolic acids and application to Swertia species: Optimization of triterpenic acids extraction and pre-column derivatization using response surface methodology

Oleanolic acid (OA) and ursolic acid (UA) are the ubiquitous triterpenic acids in plant kingdom and have multiple biological effects. In the present study, a highly sensitive and selective method using a dual-sensitive probe 2-(5-benzoacridine)ethyl-p-toluenesulfonate (BAETS) as pre-column labeling reagent has been developed for rapid determination of the triterpenes OA and UA by HPLC with fluorescence detection (FLD) and online mass spectrometry identification. Response surface methodology as an efficient tool was employed to optimize the ultrasonic-assisted extraction of triterpenic acids from Swertia plants and the pre-column derivatization reaction, respectively, which ensured the highest triterpenic acids recoveries within the shortest extraction time and the sufficient labeling of the analyzed components. Fast separation of the isomers OA and UA could be achieved on a Hypersil BDS C8 column within 7 min. Both of OA and UA gave the good correlation coefficients of 0.9999. This developed method offered the satisfactory detection limits of 1.10 and 1.30 ng mL −1 for UA and OA, respectively. When applied to Swertia species, it showed good reproducibility.

Automated precolumn derivatization system for analyzing physiological amino acids by liquid chromatography/mass spectrometry

An automated method for high-throughput amino acid analysis, using precolumn derivatization high-performance liquid chromatography/electrospray mass spectrometry (HPLC/ESI-MS), was developed and evaluated. The precolumn derivatization step was performed in the reaction port of a home-built auto-sampler system. Amino acids were derivatized with 3-aminopyridyl-N-hydroxysuccinimidyl carbamate, and a 3 microm Wakosil-II 3C8-100HG column (100 x 2.1 mm i.d.) was used for separation. To achieve a 13 min cycle for each sample, the derivatization and separation steps were performed in parallel. The results of the method evaluation, including the linearity, and the intra- and inter-precision, were sufficient to measure physiological amino acids in human plasma samples. The relative standard deviations of typical amino acids in actual human plasma samples were below 10%.

Determination of Amitrole in Environmental Water Samples with Precolumn Derivatization by High-Performance Liquid Chromatography

Amitrole is a nonselective polar herbicide that can easily pollute ground and surface waters because of its high solubility in water. A precolumn derivatization high-performance liquid chromatographic method for amitrole analysis has been developed. Derivatization of amitrole was performed with 4-chloro-3,5-dinitrobenzotrifluoride (CNBF). The derivatization conditions and the influence of elution composition on the separation were investigated. In pH 9.5 H(3)BO(3)-Na(2)B(4)O(7) media, the reaction of amitrole with CNBF was complete at 60 degrees C after 30 min. The stability of the derivative under light irradiation and room temperature in methanol-water samples was demonstrated. The derivatized amitrole was separated on a K C(18) column (250 mm x 4.6 mm, 5 microm) at room temperature, and UV detection was applied at 360 nm. The separation of derivatized amitrole was achieved within 18 min by gradient elution mode. The method correlation coefficient was 0.9995, in concentrations ranging from 1.59 to 318 mg L(-1). The detection limit of amitrole was 0.16 mg L(-1) with a signal-to-noise ratio of 3. The proposed method was applied to the quantitative determination of amitrole in environmental water with recoveries of 92.0-103.0% and RSDs of 2.22-6.26, depending on the sample investigated. This method has good accuracy and repeatability that can be used to quantify amitrole in environmental water.

Determination of total retronecine esters‐type hepatotoxic pyrrolizidine alkaloids in plant materials by pre‐column derivatization high‐performance liquid chromatography

A pre-column derivatization high-performance liquid chromatography method with diode array detection was developed and validated to determine the total retronecine esters-type hepatotoxic pyrrolizidine alkaloids (RET-HPAs) in herbs. The RET-HPAs reacted with o-chloranil in methanolic solution heated for 3 h, and an oxidative derivative was produced that could be detected at a maximal absorption of 223 nm. The analysis was performed using a C18 column with an isocratic elution of methanol and aqueous 0.01% triethylamine (adjusted to pH 4 with formic acid), and the detection was carried out with DAD at 223 nm. The validation of the method included linearity, sensitivity, recovery and stability. It showed a good linear regression (r(2) > 0.9900) in the range of 2.5-250 microM with a limit of detection (S/N = 3) of 0.5 microM. The method provided desirable repeatability with overall intra- and inter-day variations of less than 4.6%. The obtained recoveries for both of the extraction and derivatization process were between 94.6 and 100.7% (n = 3).

Residue determination of glyphosate in environmental water samples with high-performance liquid chromatography and UV detection after derivatization with 4-chloro-3,5-dinitrobenzotrifluoride

A pre-column derivatization high-performance liquid chromatographic method for glyphosate analysis has been developed. Derivatization of glyphosate was performed with 4-chloro-3,5-dinitrobenzotrifluoride (CNBF). In pH 9.5 H 3BO 3–Na 2B 4O 7 media, the reaction of glyphosate with CNBF completed at 60 °C for 30 min. The labeled glyphosate was separated on a Kromasil C18 column (250 mm × 4.6 mm, 5 μm) at room temperature and UV detection was applied at 360 nm. The separation of labeled glyphosate was achieved within 15 min by gradient elution mode. Compared to other pre-column derivatization, this derivatization was performed more mildly, the derivative was more stable, and the detection limits of a few reagents were higher than CNBF, except 9-fluorenylmethyl chloroformate (FMOC-Cl) using fluorescence and mass spectrometry, however, this reagent avoid to be removed after derivatization like FMOC-Cl. The detection limit of glyphosate was 0.009 mg L −1 (S/N = 3) without preconcentration and reach MRL, which is set at the level of 0.1 mg L −1 in China. The method linearity correlation coefficient was 0.9999, in concentrations ranging from 0.3 to 48.5 mg L −1. The proposed method has been applied to the quantitative determination of glyphosate in environmental water with recoveries of 91.80–100.20% and R.S.D. of 2.27–6.80, depending on the sample investigated.

Pre-column Derivatization High Performance Liquid Chromatography Tandem Mass Spectrometric Determination of Trace Level of Amino Acids in Rat Serum

A simple and sensitive method based on the derivatization of 20 amino acids with 1,2-benzo-3,4-dihydrocarbazole-9- ethyl chloroformate (BCEOC) as derivatization reagent on a reversed-phase Hypersil BDS C 18 column (4.6 mm × 200 mm, 5 μm) with a gradient elution followed by high performance liquid chromatography (HPLC) via fluorescence detection at 390 nm (excitation wavelength of 333 nm) and tandem mass spectrometric identification has been developed. Optimum derivatization, giving the corresponding stable fluorescent derivatives, was obtained by reacting amino acids with BCEOC at 40 ° for 10 min in borate buffer (pH 9.0) with four times excess of molar reagent. The linear range of 20 amino acids were 51.6 fmol–105.6 pmol, all correlation coefficients >0.9995, detection limits were 6.3–177.6 fmol (at signal to noise 3:1, S/N=3:1). The identification of amino acid derivatives was carried out by post-column tandem mass spectrometry with electrospray ion (ESI) source, and the MS/MS cleavage mode of representative tyrosine derivative was analyzed. Under all the above optimum experimental conditions, the contents of 20 amino acids in rat serum of 3 groups (A: Quiet, B: At exercising exhaust, C: 12 h after exercising exhaust) were determined. It was indicated that the contents of 20 amino acids in group B were obviously higher than those in group A, and the contents of amino acids of group C and group A were almost the same. The established method exhibited high sensitivity and excellent reproducibility, and provided a new technology for the determination of amino acids in rat serum.

Determination of biogenic amines in apples and wine with 8-phenyl-(4-oxy-acetic acid N-hydroxysuccinimide ester)-4, 4-difluoro-1,3,5,7-tetramethyl-4-bora-3a,4a-diaza- s-indacene by high performance liquid chromatography

A precolumn-derivatization high performance liquid chromatographic method for biogenic amine analysis has been developed. Derivatization of biogenic amines was performed with a new fluorescent reagent, 8-phenyl-(4-oxy-acetic acid N-hydroxysuccinimide ester)-4,4-difluoro-1,3,5,7-tetramethyl-4-bora-3a,4a-diaza- s-indacene (TMPAB-OSu), which was synthesized in our lab. The derivatization conditions and the influence of elution composition on the separation were investigated. In pH 8.5 H 3BO 3–Na 2B 4O 7 media, the reaction of biogenic amines with TMPAB-OSu completed at 45 °C for 5 min. The labeled biogenic amines were separated on an ZORBAX Eclipse XDB-C 8 column (150 mm × 4.6 mm, 5 μm) and detected with fluorescence at excitation and emission wavelengths of 497 and 509 nm, respectively. Detection limits of biogenic amines were 0.1–4 nmol L −1, at a signal-to-noise ratio of 3. The proposed method has been applied to the quantitative determination of spermine, phenethylamine, spermidine, cadaverine and putrescine in pericarp and pulp of mature and immature apples and wine with recoveries of 95–102% and R.S.D. of 0.7–3.9.

Monitoring of 8-oxo-7,8-dihydro-2′-deoxyguanosine in urine by high-performance liquid chromatography after pre-column derivatization with glyoxal reagents

A new, simple and sensitive pre-column fluorescence derivatization high-performance liquid chromatographic method for the determination of the oxidative DNA stress marker, 8-oxo-7,8-dihydro-2'-deoxyguanosine, was developed. Solid-phase extraction using an Oasis HLB cartridge avoided troublesome sample preparation steps, interference from charged species and frequent and essential electrode maintenance in electrochemical procedures. 8-Oxo-7,8-dihydro-2'-deoxyguanosine and other guanine compounds were selectively derivatized with glyoxal reagents (phenylglyoxal, 3,4-methylenedioxyglyoxal, 2-naphtylglyoxal and 6-methoxynaphthylglyoxal) at 40-60 degrees C. Derivatization with 6-methoxynaphthylglyoxal at 40 degrees C for 30 min gave the strongest fluorescence product. The fluorescence derivatives from reaction with 6-methoxynaphthylglyoxal were separated on a Capcell Pak C18 SG 120A column (4.6 mm i.d. x 150 mm, 5 microm) with acetonitrile-5 mM phosphate buffer (pH 6.0; 3:7, v/v) as mobile phase. The detection wavelength of the fluorescence derivative of 8-oxo-7,8-dihydro-2'-deoxyguanosine was lambda(ex) 400 nm and lambda(em) 510 nm. The detection limit of 8-oxo-7,8-dihydro-2'-deoxyguanosine was 1 ng/mL using 50 mL of urine. The calibration graphs were linear up to 30 microg/mL for 8-oxo-7,8-dihydro-2'-deoxyguanosine. The relative standard deviation of 20 ng/mL of 8-oxo-7,8-dihydro-2'-deoxyguanosine was 7.0%. The proposed method was compared with the enzymatic ELISA 8-oxo-7,8-dihydro-2'-deoxyguanosine analysis method (8-OH-dG Check, JaICA, Shizuoka, Japan). The correlation coefficient was 0.79 (n = 20) and y = 0.85x + 5.34. The proposed method was applied to the monitoring of 8-oxo-7,8-dihydro-2'-deoxyguanosine in urine from male heavy smokers.

Determination of fluoxetine enantiomers in rat plasma by pre-column fluorescence derivatization and column-switching high-performance liquid chromatography.

A column-switching HPLC method employing both octadecylsilica (ODS) and chiral columns with fluorescence detection was developed for the determination of enantiomer of fluoxetine (FLX), an antidepressant drug, in rat plasma. Racemic FLX was derivatized with a fluorescent reagent, 4-(N-chloroformylmethyl-N-methyl)amino-7-nitro-2,1,3-benzoxadiazole (NBD-COCl) or 4-(N-chloroformylmethyl-N-methyl)amino-7-N,N-dimethylaminosulfonyl-2,1,3-benzoxadiazole (DBD-COCl) and the enantiomeric separation of the resultant derivatives was examined on an amylose-based chiral column (CHIRALPAK AD-RH) in the reversed-phase mode. The derivative with NBD-COCl (NBD-FLX) showed a sufficient separation factor (a) and resolution (Rs) compared with that with DBD-COCl. Thus, FLX was derivatized with NBD-COCl and the resultant NBD-FLX was first quantified on the ODS column and then introduced to the CHIRALPAK AD-RH column via a six-port switching valve to examine the enantiomeric ratio. The intra- and inter-day accuracy (97.6-112.7%) and precision (1.47-10.60%) were satisfactory in the range 10-1000 nM FLX and the limit of quantification was approximately 10 nM. The absolute recoveries of FLX with hexane from rat plasma were in the range 87.5-92.2% (n = 3). The method was applied to determine FLX enantiomers in the plasma of rats administered FLX orally, and it was shown that the R-isomer was eliminated faster than the S-isomer.

Determination of sucralose in foods by HPLC using pre-column derivatization

The development of a sensitive pre-column derivatization high-performance liquid chromatography (HPLC) method for determination of sucralose is reported. Sucralose is converted into a strongly ultraviolet (UV)-absorbing derivative, possessing strong absorption at 260 nm, by treatment with p-nitrobenzoyl chloride (PNBCl). Homogenized samples were dialyzed and washed with a Bond Elut ENV cartridge, then the eluate was evaporated to dryness and the residue was derivatized. Subsequently, the sucralose derivative was purified with hexane-ethyl actate (9:1) in a silica cartridge, and then the sucralose derivative was eluted with acetone. HPLC was performed on a phenyl column, using acetonitrile-water (73:27) as a mobile phase with UV detection (260 nm). The calibration curve was linear in the range of 1 microgram/mL to 50 micrograms/mL of sucralose. The recoveries of sucralose from eight kinds of foods spiked at the levels of 0.20 and 0.05 g/kg of sucralose were more than 76.2% with SD values in the range from 0.90% to 4.31%. The quantitative limit of the developed method was 0.005 g/kg for sucralose in samples.

Pre-column derivatization high-performance liquid chromatographic method for determination of cysteine, cysteinyl–glycine, homocysteine and glutathione in plasma and cell extracts

A sensitive high-performance liquid chromatographic method for quantification of sulphydryl and disulfide amino acids in human plasma using ultra violet spectrophotometric detection was developed. Precolumn derivatization with 5,5′-dithio-bis-nitrobenzoic acid (DTNB) and an optional pre-derivatization reaction with dithiothreitol allowed both quantitative reduction of disulfides for measurement of total amino acid levels and the measurement of the reduced forms. A dynamic range of 500 nmol/l–750 μmol/l allowed the major analytes of interest to be quantified in plasma without sample dilution. The assay is a sensitive and precise method for the determination of sulphydryl and disulfide amino acids in plasma and cell extracts.

A precolumn derivatization high-performance liquid chromatographic method with improved sensitivity and specificity for the determination of astragaloside IV in Radix Astragali.

A reversed-phase high-performance liquid chromatographic method is developed for the determination of astragaloside (AGS) IV, which is known as the active constituent of Radix Astragali. The method uses precolumn derivatization with benzoyl chloride to form the benzoyl ester of AGS IV quantitatively and is carried out with a wide-ranging concentration (0.004-0.080 mg/mL) of the derivatized AGS IV. The eluent consists of 90% methanol, 4% tetrahydrofuran, 6% water, and 0.2% triethylamine, with vitamin D3 added as the internal standard.