Determination of fluvoxamine in rat plasma by HPLC with pre-column derivatization and fluorescence detection using 4-fluoro-7-nitro-2,1,3-benzoxadiazole

A sensitive, simple and reliable method using high-performance liquid chromatographic (HPLC) assay of fluvoxamine (FLU), a selective serotonin reuptake inhibitor (SSRI), in rat plasma after pre-column derivatization with 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F) was developed in this study. Extracted plasma samples were mixed with NBD-F at 60 degrees C for 5 min and injected into HPLC. Retention times of FLU and an internal standard (propafenone) derivative were 15.5 and 13.5 min, respectively. The calibration curve was linear over the range 0.015-1.5 microg/mL (r2 = 0.9985) and the lower limits of detection and quantification of FLU were 0.008 and 0.015 microg/mL, respectively, in 100 microL of plasma. The derivative sample was stable at 4 degrees C for 1 day. The coefficients of variation for intra-day and inter-day assay of FLU were less than 8.3 and 9.6%, respectively. Other SSRIs and centrally acting drugs did not interfere with the peak of the FLU derivative. The method was applied for analysis of the plasma samples from rats treated with FLU. These results indicate that the method presented is useful to determine the FLU levels in rat plasma of volumes as small as 100 microL and can be applied to pharmacokinetic studies.     

Sensitive determination of 4-(4-chlorophenyl)-4-hydroxypiperidine, a metabolite of haloperidol, in a rat biological sample by HPLC with fluorescence detection after pre-column derivatization using 4-fluoro-7-nitro-2,1,3-benzoxadiazole

4-(4-Chlorophenyl)-4-hydroxypiperidine (CPHP), one of the metabolites of haloperidol, is considered to exhibit brain toxicity. CPHP concentrations in plasma and tissue homogenates (each 200 microL) from rats were analyzed by HPLC fluorescence detection after pre-column derivatization with 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F). After basic extraction of the samples with benzene, the derivatization with NBD-F was conducted in borate buffer (pH 8.0) at 60 degrees C for 3 min. Mexiletine was carried through the procedure as an internal standard. The regression equation for CPHP showed a good linearity in the range of 0.03-1 microg/mL with a detection limit of 0.008 microg/mL. The coefficient of variation was less than 11.6%. Plasma concentration-time courses of CPHP after intraperitoneal or per oral administration of CPHP, haloperidol or reduced haloperidol were examined, and the pharmacokinetic parameters were estimated. Additionally, CPHP levels in various tissues at 8 h after intraperitoneal administration of these compounds were compared. The method was simple and sensitive, useful for determination of CPHP in rat biological samples using as little as 200 microL of sample volume and could be applied for pharmacokinetic study.     

Simultaneous determination of amantadine and rimantadine by HPLC in rat plasma with pre-column derivatization and fluorescence detection for pharmacokinetic studies

We investigated simultaneous high-performance liquid chromatographic (HPLC) determination of amantadine hydrochloride (AMA) and rimantadine hydrochloride (RIM) levels in rat plasma after fluorescent derivatization with o-phthalaldehyde and 2-mercaptoethanol. Afterwards, the method was applied to determine their pharmacokinetics. The retention times of AMA and RIM derivatives were 12.6 and 22.2 min and the lower limits of detection were 0.025 and 0.016 microg/mL, respectively. The coefficients of variation for intra- and inter-day assay of AMA and RIM were less than 5.1 and 7.6%, respectively. After i.v. administration of AMA or RIM to rats, the total body clearance and distribution volume at the steady-state of RIM were higher than those of AMA. Bioavailability of AMA and RIM was 34.9 and 37.2%, respectively. When AMA and RIM were p.o. co-administered, the area under the plasma concentration--time curve of RIM was significantly lower than that after RIM alone. On the other hand, pharmacokinetic parameters of AMA did not significantly change. These results indicate that our HPLC assay is simple, rapid, sensitive and reproducible for simultaneously determining AMA and RIM concentrations in rat plasma and is applicable to their pharmacokinetic studies. Also, co-administration of AMA and RIM may result in the lack of pharmacological effects of RIM.     

Simultaneous determination of homocysteine,methionine and cysteine in maternal plasma after delivery by HPLC‐fluorescence detection with DBD‐F as a label

An HPLC‐fluorescence (FL) method for determination of sulfur‐containing amino acids such as homocysteine (Hcy), methionine (Met) and cysteine (Cys) in human plasma was developed. The sulfur‐containing amino acids were labeled with 4‐(N,N‐dimethylaminosulfonyl)‐7‐fluoro‐2,1,3‐benzoxadiazole (DBD‐F). Calibration curves in the range of 1–100 µm (Hcy and Met) and 5–500 µm (Cys) indicated good linearities (r ≥ 0.998). The limits of detection at a signal‐to‐noise ratio of 3 were 0.13 (Hcy), 0.02 (Met) and 0.11 µm (Cys), respectively. Acceptable results for accuracy and precision of intra‐ and inter‐day measurements were obtained. The results of Hcy and Cys obtained by the proposed method indicated good correlations with the conventional method (r > 0.911, n = 20). Furthermore, the method was applied to determination of the sulfur‐containing amino acids in maternal plasma (n = 200) after delivery. The concentrations of Hcy, Met and Cys as a median (inter quartile range, Q₁ and Q₃) were 5.37 (3.32–7.79) μm , 25.20 (20.10–31.06) μm and 147.25 (102.81–189.31) μm , respectively. Copyright © 2012 John Wiley & Sons, Ltd.     

Determination of catecholamines and serotonin by micellar electrokinetic chromatography with laser-induced fluorescence detection

6-Oxy-(N-succinimidyl acetate)-9-(2'-methoxycarbonyl) fluorescein, a new synthesized fluorescent reagent, was established for the first time as a label for the sensitive analysis of catecholamines (CAs) and serotonin (5-HT) by micellar electrokinetic chromatography (MEKC) with laser-induced fluorescence detection. After careful study on the derivatization conditions such as pH value, reagent concentration, temperature and reaction time, the labeling reaction was accomplished as quickly as 7 min with stable yield. The separation parameters for the CAs and 5-HT were also optimized in detail. The derivatives were baseline separated in a running buffer containing 30 mM boric acid and 15 mM sodium dodeculsulfate at pH 9.0. The detection limits ranged from 5 x 10(-10) to 2 x 10(-9) M (signal-to-noise ratio = 3). The rapid and sensitive method was also applied to the determination of the CAs and 5-HT of urine samples.     

柱前衍生反相高效液相色谱-荧光检测法测定大鼠血浆中的奈替米星

建立了一种简便、灵敏的氯甲酸芴甲酯(FMOC-Cl)柱前衍生反相高效液相色谱-荧光检测血浆中奈替米星的新方法,同时研究了其药代动力学。对色谱条件进行了优化,采用ZORBAX Eclipse XDB-C8柱(150 mm×4.6 mm,5 μm),流动相为乙腈-水(体积比为85:15),流速为1.0 mL/min,荧光检测激发波长为265 nm,发射波长为315 nm,得到奈替米星的平均加标回收率为96.62%～100.84%(n=3),对奈替米星检测的线性范围为0.045～8.88 mg/L,相关系数为0.9993,方法的日内与日间精密度分别低于3%与3.5%,最低检出限(S/N=3)与定量限(以3倍检出限计)分别为0.01和0.03 mg/L。方法简便、快速、灵敏,样品用量少(30 μL奈替米星血浆溶液已能满足该药含量的测定以及药物代谢的研究),为大鼠体内奈替米星的药代动力学研究提供了可靠的分析手段。     

Determination of kynurenine levels in rat plasma by high-performance liquid chromatography with pre-column fluorescence derivatization

Kynurenine (KYN), a tryptophan metabolite, is a crucial compound for modulating neurotransmission because it can be metabolized in vivo into both quinolinic acid and kynurenic acid, which are the agonist and antagonist, respectively, of N-methyl-d-aspartate receptor. For the highly sensitive detection of KYN by high-performance liquid chromatography (HPLC), a fluorescence derivatization of KYN with a benzofurazan-type fluorogenic reagent, 4-N,N-dimethylaminosulfonyl-7-fluoro-2,1,3-benzoxadiazole (DBD-F) was investigated in the present study. KYN was derivatized with DBD-F (DBD-KYN) at 60 °C for 30 min, and separated on an octadecylsilica column with a gradient elution of the mobile phase, which consists of 0.1% formic acid in acetonitrile/methanol/water. DBD-KYN was detected fluorimetrically at 553 nm with an excitation wavelength of 431 nm. The limits of detection and quantification were approximately 0.30 pmol [signal-to-noise ratio (S/N) 3] and 1.0 pmol (S/N, 10) on column, respectively. Plasma KYN levels were successfully determined using 10 μL of rat plasma with satisfactory precision and accuracy. Intra- and inter-day precisions and accuracies were 1.7-6.8%, and −10 to 9.6%, respectively. KYN levels in plasma of male Sprague-Dawley rats (7 weeks old) were approximately 2.4 ± 0.32 μmol L⁻¹ (n = 4). The proposed HPLC method was applied to determine KYN levels in the plasma of ketamine-treated rats—the animal model of schizophrenia.     

Determination of methyl methanesulfonate and ethyl methanesulfonate in methanesulfonic acid by derivatization followed by high‐performance liquid chromatography with ultraviolet detection

Methanesulfonic acid is routinely used in pharmaceuticals but can contain potentially genotoxic impurities such as methyl methanesulfonate and ethyl methanesulfonate. The aim of this study was to develop a simple high‐performance liquid chromatography with ultraviolet detection method for determining methyl methanesulfonate and ethyl methanesulfonate in methanesulfonic acid. Samples (250 mg) in water/acetonitrile (200 μL) were first combined with 10.0 mol/L sodium hydroxide solution (270 μL). Then they were mixed with 2.0 mg/mL N ,N‐diethyldithiocarbamate (500 μL), diluted to 5 mL with N ,N‐dimethylacetamide and allowed to react at 80°C for 1 h. The derivatives were analyzed using gradient high‐performance liquid chromatography with ultraviolet detection (277 nm) and structurally elucidated by liquid chromatography with mass spectrometry. With acetonitrile/5 mmol/L ammonium acetate solution as the eluent and 1 mL/min as the flow rate on a C18 column, the derivatives were eluted at 10.6 and 14.8 min. Good linearity (correlation coefficients > 0.999) and low limits of quantitation (0.6 ppm) were obtained. The recoveries were in the range of 80–115% with relative standard deviation < 5.0%. Finally, the established method was successfully used for the determination of methyl methanesulfonate and ethyl methanesulfonate in methanesulfonic acid.     

Enantiomeric separation of D,L-tryptophan and D,L-kynurenine by HPLC using pre-column fluorescence derivatization with R(-)-DBD-PyNCS

The enantiomeric separation of d ,l ‐tryptophan (Trp) and d ,l ‐kynurenine (KYN) was investigated by high‐performance liquid chromatography using pre‐column fluorescence derivatization with a chiral fluorescent labeling reagent, R(−)‐4‐(3‐isothiocyanatopyrrolidin‐1‐yl)‐7‐ (N,N‐dimethylaminosulfonyl)‐2,1,3‐benzoxadiazole [R(−)‐DBD‐PyNCS]. Using an octadecylsilica column, namely, an Inertsil ODS‐3 column (250 × 2.0 mm; i.d., 3 µm), four fluorescence peaks of D‐ and l ‐Trp as well as d ‐ and l ‐KYN derivatized with R(−)‐DBD‐PyNCS were clearly observed, and their chemical structures were confirmed by HPLC–time‐of‐flight–mass spectrometry. Simultaneous separation was achieved under the mobile phase condition of 1.5% acetic acid in H₂O–CH₃CN (60:40), and the separation factors of d ,l ‐Trp and d ,l ‐KYN derivatized with R(−)‐DBD‐PyNCS were 1.22 and 1.19, respectively. Fluorescence detection was carried out by setting the emission wavelength at 565 nm, and the excitation wavelength at 440 nm, and the detection limits were approximately 0.3–0.5 pmol (signal‐to‐noise ratio of 3). Copyright © 2010 John Wiley & Sons, Ltd.     

Sensitive determination of MDMA and its metabolite MDA in rat blood and brain microdialysates by HPLC with fluorescence detection

Simultaneous determination of 3,4-methylenedioxymethamphetamine (MDMA) and 3,4-methylenedioxyamphetamine (MDA) in rat blood and brain microdialysates by high-performance liquid chromatography with fluorescence detection (HPLC-FL) was developed. Microdialysates were directly subjected to derivatization with 4-(4,5-diphenyl-1H-imidazol-2-yl)benzoyl chloride (DIB-Cl). The DIB-derivatives of MDMA, MDA and the internal standard, 1-methyl-3-phenylpropylamine (MPPA), were isocratically separated on an ODS column using a mixture of 50 mm phosphate buffer (pH 7.0)-acetonitrile-methanol-2-propanol (50:45:5:2, v/v/v/v %) as an eluent at a flow rate of 1.5 mL/min. The calibration curves of MDA and MDMA spiked to blood and brain microdialysates were linear over the ranges 2.5-500 and 5.0-1000 ng/mL, respectively. The detection limits of MDA and MDMA were 1.2 and 4.2 for blood and 1.3 and 4.8 ng/mL for brain, respectively. Additionally, the intra- and the inter-assay precisions were lower than 5.6% for the blood and brain microdialysates (n = 4). The proposed method was successfully applied for the monitoring of MDMA and its metabolite MDA in rat blood and brain microdialysates, and the pharmacokinetic parameters of MDMA and MDA in the microdialysates after administration of MDMA (5 mg/kg, i.p.) with or without caffeine (20 mg/kg, i.p.) were evaluated.     

Determination of calycosin-7-O-beta-D-glucopyranoside in rat plasma and urine by HPLC

A reversed-phase high-performance liquid chromatographic (HPLC) assay for calycosin-7-O-beta-D-glucopyranoside in rat plasma and urine with solid-phase extraction (SPE) was developed. Rutin was employed as an internal standard. The mobile phase consisted of acetonitrile-water (16:84, v/v) at a flow rate of 1.0 mL/min. Detection was set at 280 nm. The limit of quantitation of calycosin-7-O-beta-D-glucopyranoside was 0.2 microg/mL in both plasma and urine. The standard curve was linear from 0.2 to 10.0 microg/mL in plasma, and 0.2 to 5.0 microg/mL in urine. Both intra- and inter-day precision of the calycosin-7-O-beta-d-glucopyranoside were determined and their RSD did not exceed 10%. The method was successfully applied to the analysis of samples obtained from a basic pharmacokinetic study, in which calycosin-7-O-beta-d-glucopyranoside was administered orally to rats.     

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.     

An improved method for proteomics studies in C. elegans by fluorogenic derivatization, HPLC isolation, enzymatic digestion and liquid chromatography--tandem mass spectrometric identification

An improved method for proteomics studies, which includes the fluorogenic derivertization of protein mixtures with 7-chloro-4-(dimethylaminoethylaminosulfonyl)-2,1,3-benzoxadiazole (DAABD-Cl), followed by HPLC isolation, enzymatic digestion and identification of the derivatized proteins by HPLC-electrospray ionization (ESI)-MS/MS with the probability-based protein identification algorithm, identified 103 proteins in the soluble extract (10 microg protein) of Caenorhabditis elegans.     

Analysis of primary aromatic amines using precolumn derivatization by HPLC fluorescence detection and online MS identification

2-(2-phenyl-1H-phenanthro-[9,10-d]imidazole-1-yl)-acetic acid (PPIA) and 2-(9-acridone)-acetic acid (AAA), two novel precolumn fluorescent derivatization reagents, have been developed and compared for analysis of primary aromatic amines by high performance liquid chromatographic fluorescence detection coupled with online mass spectrometric identification. PPIA and AAA react rapidly and smoothly with the aromatic amines on the basis of a condensation reaction using 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC) as dehydrating catalyst to form stable derivatives with emission wavelengths at 380 and 440 nm, respectively. Taking six primary aromatic amines (aniline, 2-methylaniline, 2-methoxyaniline, 4-methylaniline, 4-chloroaniline, and 4-bromoaniline) as testing compounds, derivatization conditions such as coupling reagent, basic catalyst, reaction temperature and time, reaction solvent, and fluorescent labeling reagent concentration have also been investigated. With the better PPIA method, chromatographic separation of derivatized aromatic amines exhibited a good baseline resolution on an RP column. At the same time, by online mass spectrometric identification with atmospheric pressure chemical ionization (APCI) source in positive ion mode, the PPIA-labeled derivatives were characterized by easy-to-interpret mass spectra due to the prominent protonated molecular ion m/z [M + H](+) and specific fragment ions (MS/MS) m/z 335 and 295. The linear range is 24.41 fmol-200.0 pmol with correlation coefficients in the range of 0.9996-0.9999, and detection limits of PPIA-labeled aromatic amines are 0.12-0.21 nmol/L (S/N = 3). Method repeatability, precision, and recovery were evaluated and the results were excellent for the efficient HPLC analysis. The most important argument, however, was the high sensitivity and ease-of-handling of the PPIA method. Preliminary experiments with wastewater samples collected from the waterspout of a paper mill and its nearby soil where pollution with aromatic amines may be expected show that the method is highly validated with little interference in the chromatogram.     

Determination of trace biogenic amines with 1,3,5,7-tetramethyl-8-(N-hydroxysuccinimidyl butyric ester)-difluoroboradiaza-s-indacene derivatization using high-performance liquid chromatography and fluorescence detection

A reversed-phase high-performance liquid chromatographic method based on chemical derivatization with fluorescence detection has been developed for analyzing biogenic amines in food and environmental samples. A BODIPY-based fluorescent reagent, 1,3,5,7-tetramethyl-8-(N-hydroxysuccinimidyl butyric ester)-difluoroboradiaza-s-indacene (TMBB-Su), was employed for the derivatization of these biogenic amines at 20 °C for 20 min in pH 7.20 borate buffer after careful investigation of the derivatization conditions including reagent concentration, buffer solution, reaction temperature and reaction time. Separation of biogenic amines with gradient elution was conducted on a C8 column with methanol-tetrahydrofuran-water as mobile phase. The detection limits were obtained in the range from 0.1 to 0.2 nM (signal-to-noise=3). This procedure has been validated using practical samples. The study results demonstrated a potential of employing high-performance liquid chromatography (HPLC) with 1,3,5,7-tetramethyl-8-(N-hydroxysuccinimidyl butyric ester)-difluoroboradiaza-s-indacene labeling as a tool for quantitative analysis of biogenic amines involved in various matrices.     

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。     

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.     

On-capillary derivatization and analysis of amino acids in human plasma by capillary electrophoresis with laser-induced fluorescence detection: application to diagnosis of aminoacidopathies

A capillary electrophoresis with laser-induced fluorescence detection method for the analysis of free amino acids (AA) in human plasma was developed. A mixture of 16 AA was on-capillary derivatized with 3-(2-furoyl)quinoline-2-carboxaldehyde (FQ) and separated inside the capillary in less than 30 min using 70 mM borax-3.5 mM SDS pH 9.3 as running buffer. Four plasma samples from a healthy donor and patients suffering from phenylketonuria, propionic acidemia, and tyrosinemia type II were studied. Repeatabilities calculated as intra-day RSD (n = 3) values for the AA involved in these aminoacidopathies (glycine, phenylalanine, and tyrosine) were in the range of 0.3 to 1.2% for migration time and 3.7 to 8.2% for peak height. Reproducibilities calculated as inter-day RSD (n = 4) values for the same AA were between 0.7 and 1.4% for migration time and 4.7 and 9.1% for peak height. A fast qualitative analysis allowed the identification of the corresponding disease by comparing the electrophoretic profiles from the patient and the healthy donor and noting the increased level of the specific AA accompanying each individual disease. The results of the quantitative analysis for glycine, phenylalanine, and tyrosine in the plasma samples studied using the developed method showed a good agreement with those provided by the Center of Diagnosis of Molecular Diseases using a standard method for AA analysis.     

Determination of pamidronate in human whole blood and urine by reversed-phase HPLC with fluorescence detection

Pamidronate is a bisphosphonate that is effective in treating bone disease including osteopenia and osteoporosis in adults. A sensitive and reliable method for the analysis of pamidronate in whole blood and urine is key to the development of this drug for use in children. A previously described method for pamidronate analysis serum and urine did not consistently detect the drug at satisfactory levels in whole blood. The procedure involves co-precipitation of the bisphosphonates with calcium phosphate, pre-column derivitization with fluorescamine, HPLC utilizing a Nucleosil C(18) column, and fluorescence detection with excitation at 395 nm and emission at 480 nm.Changes to the original protocol included the use of a new internal standard (alendronate), the optimization of the concentration of ethylenediaminetetraacetic acid (EDTA) for dissolving the precipitate, and the elimination of the acidification step prior to deproteinization. The optimum EDTA concentration, which had a significant effect on the labeling capability of fluorescamine, was determined to be 20 mm.A good separation between pamidronate and alendronate was achieved using a heated (40 degrees C ) Nucleosil C(18), 10 micro m particle size column. The mobile phase was an aqueous solution of 1 mm Na(2)EDTA-methanol (97:3, v/v) adjusted to pH 6.5 using a fl ow-rate of 1 mL/min. Fluorescence detection was set at 395 nm for excitation and at 480 nm for emission. The limit of quantitation for pamidronate was 0.5 micro g/mL in whole blood and 0.1 micro g/mL in urine. The method was applied to both whole blood and urine samples from pediatric patients.     

New derivatizing reagent for analysis of diethylene glycol by HPLC with fluorescence detection

N‐(2‐Phenyl‐indolyl)‐acetic acid (PIAA), a new fluorescent derivatizing reagent, was used for the determination of diethylene glycol (DEG) by high‐performance liquid chromatography with fluorescence detection. DEG was derivatized to ester by using PIAA in the presence of 1‐ethyl‐3‐(3‐dimethylaminopropyl)carbodimide hydrochloride (as dehydrating agent) and 4‐(dimethylamino)pyridine (as base catalyst) in acetonitrile at 60°C for 75 min. The influence of solvent, temperature, catalyst base, concentration of labeling reagent, and couple reagent on the derivatization was investigated. The fluorescence detection was performed with excitation at 340 nm and emission at 377 nm. Baseline separation was obtained on an Ultimate XB‐C18 analytical column with water/acetonitrile gradient elution, good linearity was obtained within 0.5–50 μg/mL with a correlation coefficient of 0.9997. The limit of detection was 0.01 μg/mL (signal‐to‐noise ratio = 3). The method has been successfully applied to determine DEG in toothpaste samples with satisfactory recoveries ranging from 89.0 to 94.9%. The proposed method was shown to be a promising technique for the determination of DEG with high sensitivity.