Reversed‐phase liquid chromatographic determination of enantiomers of atenolol in rat plasma using derivatization with Marfey's reagent

An HPLC method was established for enantioseparation of (R,S)‐atenolol (ATE) and determination of enantiomers in rat plasma. Marfey's reagent (1‐fluoro‐2,4‐dinitrophenyl‐5‐L‐alanine amide, FDNP‐L‐Ala‐NH₂, MR) was used as chiral derivatizing reagent with detection of diastereomers at 340 nm. It was shown that the R‐isomer eluted before the S‐isomer. The method was validated for linearity, repeatability, limits of detection and limit of quantification (LOQ). Recovery of ATE at LOQ was 92.8% for (R)‐ATE and 92.6% for (S)‐ATE. Copyright © 2009 John Wiley & Sons, Ltd.     

Indirect resolution of enantiomers of penicillamine by TLC and HPLC using Marfey's reagent and its variants

TLC and HPLC methods were developed for indirect chiral separation of penicillamine (3,3-dimethylcysteine) enantiomers after derivatization with Marfey's reagent (FDNP-Ala-NH(2)) and two of its structural variants, FDNP-Phe-NH(2) and FDNP-Val-NH(2). The binary mobile phase of phenol-water (3:1 v/v) and solvent combinations of acetonitrile and triethylamine phosphate buffer were found to give the best separation in normal and reversed-phase TLC, respectively. The diastereomers were also resolved on a reversed-phase C18 HPLC column with gradient elution of acetonitrile and 0.01 m trifluoroacetic acid. The results due to these three reagents were compared. The method was successful for checking the enantiomeric impurity of l-penicillamine in d-penicillamine and to check the enantiomeric purity of pharmaceutical formulations of d-penicillamine. The method was validated for linearity, repeatability, limit of detection and limit of quantification.     

Indirect enantioresolution of (R,S)‐mexiletine by reversed‐phase high‐performance liquid chromatography via diastereomerization with [(S,S)‐O,O'‐di‐p‐toluoyl tartaric acid anhydride], (S)‐naproxen and nine chiral reagents synthesized as variants of Marfey's reagent

Eleven chiral derivatizing reagents (CDRs) were used for preparation of diastereomers of (R,S)‐mexiletine containing a primary amino group in close proximity to the stereogenic center. One anhydride, namely [(S,S)‐O,O'‐di‐p‐toluoyl tartaric acid anhydride] was synthesized and (S)‐naproxen was used as such as the chiral derivatizing reagent. The other nine CDRs were synthesized by substituting one of the fluorine atoms in 1,5‐difluoro‐2,4‐dinitrobenzene with six amino acid amides and three amino acids. The diastereomers were separated by reversed‐phase high‐performance liquid chromatography. The method was validated for linearity, accuracy, limit of detection and limit of quantification. The limit of detection was found in the range of 10–30 pmol. Copyright © 2010 John Wiley & Sons, Ltd.     

柱前手性荧光衍生反相高效液相色谱法分离甲状腺素对映体

以R(-)-4-N,N-二甲基磺酰胺-7-(3-异氰酸吡咯烷)-2,1,3-苯并氧杂咪唑（R(-)-DBD-PyNCS）为手性荧光衍生化试剂,成功地拆分了甲状腺素对映体D,L-四碘甲状腺原氨酸（T4）和L-三碘甲状腺原氨酸(T3)。在反应温度为40 ℃、反应时间为20 min时,R(-)-DBD-PyNCS在碱性介质中可与甲状腺素对映体生成稳定的非对映体衍生物。该衍生物在以乙腈-水-醋酸(体积比为60∶40∶1)为流动相,流速为1.0 mL/min,色谱柱为Intersil-ODS-3 C18柱(150 mm×4.6 mm,5 μm)的色谱条件下得到了充分的分离。采用荧光检测器在激发波长460 nm、发射波长550 nm下检测。D,L-T4和L-T3分别在0.016～0.30 μg/μL和0.0067～0.22 μg/μL范围内,峰面积与浓度呈良好的线性关系(r＞0.999)。D,L-T4和L-T3的最低检出限分别为0.02 μg/mL和0.85 μg/mL(S/N=3)。在D-T4、L-T4、L-T3质量浓度分别为0.10 μg/μL下测得峰面积的相对标准偏差分别为3.40%,1.63%,3.30%(n=7)。该方法成功地应用于甲状腺片中T4和T3的含量测定。     

Indirect reversed‐phase high‐performance liquid chromatographic and direct thin‐layer chromatographic enantioresolution of (R,S)‐Cinacalcet

Enantioresolution of the calcimimetic drug (R,S)‐Cinacalcet was achieved using both indirect and direct approaches. Six chiral variants of Marfey's reagent having l ‐Ala‐NH₂, l ‐Phe‐NH₂, l ‐Val‐NH₂, l ‐Leu‐NH₂, l ‐Met‐NH₂ and d ‐Phg‐NH₂ as chiral auxiliaries were used as derivatizing reagents under microwave irradiation. Derivatization conditions were optimized. Reversed‐phase high‐performance liquid chromatography was successful using binary mixtures of aqueous trifluoroacetic acid and acetonitrile for separation of diastereomeric pairs with detection at 340 nm. Thin silica gel layers impregnated with optically pure l ‐histidine and l ‐arginine were used for direct resolution of enantiomers. The limit of detection was found to be 60 pmol in HPLC while in TLC it was found to be in the range of 0.26–0.28 µg for each enantiomers. Copyright © 2010 John Wiley & Sons, Ltd.     

Synthesis of dinitrophenyl-l-Pro-N-hydroxysuccinimide ester and four new variants of Sanger's reagent having chiral amines and their application for enantioresolution of mexiletine using reversed-phase high-performance liquid chromatography

Four chiral derivatizing reagents (CDRs) having enantiomerically pure amines and two CDRs namely, [N-succinimidyl-(S)-2-(6-methoxynaphth-2-yl)propionate], and [dinitrophenyl-l-Pro-N-hydroxysuccinimide ester, DNP-l-Pro-SU] were synthesized and were used to prepare diastereomers of (R,S)-mexiletine (MEX); these were separated by reversed-phase high-performance liquid chromatography (RP-HPLC). The method was validated for linearity, accuracy, limit of detection (LOD) and limit of quantification (LOQ).     

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。     

在线衍生-高效液相色谱法同时测定血浆中的色氨酸及其代谢物

建立了醋酸锌在线衍生高效液相色谱法同时测定血浆中色氨酸(Trp)、犬尿氨酸(Kyn)、5-羟吲哚乙酸(5-Hiaa)和犬尿喹啉酸(Kyna)的方法。以3-硝基酪氨酸为内标(IS),采用Hypersil C-18柱(250 mm×4.0 mm, 5 μ m),以250 mmol/L醋酸锌溶液(pH 5.5)-乙腈(95:5, v/v)为流动相,流速为0.8 mL/min,柱温30℃。荧光检测波长设定:5-Hiaa为278 nm(λ_ex)/343 nm(λ_em), Kyna为244 nm(λ_ex)/400 nm(λ_em);紫外检测波长设定:Kyn和IS为360 nm, Trp为302 nm。4种物质的回收率在91.62%~114.17%之间;线性范围分别为2.50~320.00 μ mol/L(Trp), 0.32~15.36 μ mol/L(Kyn), 3.27~104.60 nmol/L(5-Hiaa), 14.00~464.80 nmol/L(Kyna);检出限分别为0.078 μ mol/L(Trp), 0.056 μ mol/L(Kyn), 0.690 nmol/L(5-Hiaa), 1.290 nmol/L(Kyna)。利用该方法对30例正常孕妇和28例女性健康志愿者的血浆进行测定,结果表明两组间Trp, Kyn和Kyna含量有显著性差异。该方法操作简便,重复性好,灵敏度高,适合于临床检测。     

Simple and rapid quantitative determination of lysinoalanine and protein hydrolysate amino acids by high-performance liquid chromatography after derivatization with dansyl chloride

Summary A rapid and simple method for the determination of both lysinoalanine (LAL) and protein hydrolysate amino acids after derivatization with dansyl chloride (5-dimethylaminoaphtalene-1sulfonyl chloride) and separation with RP-HPLC (UV detection) is presented. LAL is analysed in less than 15 minutes and complete separation of 22 amino acids is achieved in less than 30 minutes using single linear gradients of solvents (phosphate buffer and acetonitrile). Quantitative results obtained by HPLC compare well with results of the ion-exchange chromatography (amino acid analyser). The importance of the duration of the derivatization reaction and of the excess of reagent is discussed. As examples, the results of the determination of LAL in two samples of base treated α-casein and 22 samples of soy protein and the results of the analysis of amino acids in two balanced diet mixtures are presented. Presented at the 15th International Symposium on Chromatography, Nürnberg, October 1984     

纤维素-三（4-甲基苯甲酸酯）手性固定相拆分5种芳基丙酸类药物对映体及其制剂的含量测定

采用纤维素-三（4-甲基苯甲酸酯）（CTMB）手性固定相,利用反相色谱法研究了氟比洛芬、普拉洛芬、布洛芬、萘普生、洛索洛芬5种芳基丙酸类手性药物的色谱拆分行为。考察了流动相组成、酸碱添加剂及柱温对上述5种药物对映体分离的影响,并通过热力学研究及对映体结构分析对CTMB固定相的手性拆分机理进行了探讨。结果表明,除萘普生采用乙腈-0.1%（v/v）甲酸溶液外,以甲醇-0.1%（v/v）甲酸水溶液为流动相可使普拉洛芬、洛索洛芬、氟比洛芬和布洛芬的对映体间的分离度均大于1.5,CTMB固定相对这5种芳基丙酸类药物的手性拆分能力依次为普拉洛芬>洛索洛芬>氟比洛芬>布洛芬>萘普生。在各自的优化色谱条件下,将方法应用于上述5种药物制剂的含量测定,结果令人满意。     

Analysis of free amino acids in islets of Langerhans by high-performance liquid chromatography using pre-column derivatization with 4-chloro-7-nitrobenzo-2-oxa-1,3-diazole

A simple high-performance liquid chromatographic method with pre-column derivatization and fluorescence detection was developed and used for the analysis of free amino acids in islets of Langerhans; 4-chloro-7-nitrobenzo-2-oxa-1,3-diazole (NBD-Cl) served as pre-column derivatization reagent. Islets of Langerhans were separated from the pancreas of normal and obese rats, treated with pre-cooling methanol-water (80:20, v/v), and ultrasonicated to fragmentize the islets and effect deproteination. Several parameters influencing the derivatization reaction and chromatographic separation were optimized. Amino acid derivatives obtained under optimal conditions were separated on a C18 column with acetonitrile-acetate buffer as mobile phase and detected at 470 nm/540 nm (Ex/Em). Matrix effects were investigated and good linearities with correlation coefficients better than 0.9972 were obtained over a wide range of 0.42-42.11 microM for most of the amino acids. The detection limits (S/N = 3) were within the range of 6.1-51 nM. The precision of the method and recoveries were in the ranges of 1.43-10.76% (RSD%) and 85.07-108.82%, respectively. The analytical results showed that the serine content was markedly higher in normal rats than in obese rats, whereas methionine was of relatively lower content in both normal and obese rats.     

柱前衍生-反相高效液相色谱法测定不同方法煮制的猪肉及其汤汁中的游离氨基酸

建立了反相高效液相色谱(RP-HPLC)分离检测用不同方法煮制的猪肉及其汤汁中17种游离氨基酸的方法.样品经6-氨基喹啉基-N-羟基琥珀酰亚氨基甲酸酯(AQC)柱前衍生后,采用Nova- PakTMC18色谱柱分离,以AccQ·Tag Eluent A稀释液、乙腈和超纯水为流动相,梯度洗脱,检测波长为248 nm,在4...     

高效液相色谱-四极杆/静电场轨道阱高分辨质谱法测定血清中胆固醇及其6种代谢标志物

利用高效液相色谱-四极杆/静电场轨道阱高分辨质谱（HPLC-Q/Orbitrap HRMS）测定血清中的胆固醇及其6种代谢标志物（2,4-脱氢胆甾醇、7-烯胆甾醇、菜油固醇、豆固醇、β-谷固醇和角鲨烯）。以乙腈作为提取溶剂,超声提取,采用Acquity UPLC BEH C₁₈色谱柱（100 mm×2.1 mm,1.7 μm）,以95%（v/v）的甲醇-乙腈（2：8,v/v）-5%（v/v）水做流动相,等度洗脱15 min,流速0.4 mL/min。在大气压化学电离源（APCI）正离子扫描模式下,质谱采用全扫描/数据依赖二级扫描（Full MS/dd MS²）监测模式,同时获得定性和定量结果,一级分辨率为70000 FWHM,二级分辨率为17500 FWHM。7种目标物质在其线性范围内的线性相关系数（r²）均不小于0.992,检出限为0.8~62.1 μg/L,3个加标水平下的回收率为82.1%~97.5%,相对标准偏差（RSD）为1.6%~7.4%。方法准确、简单、快捷,可以作为血清中胆固醇及其6种代谢标志物的检测方法。     

Development of a sensitive fluorescent derivatization reagent 1,2-benzo-3,4-dihydrocarbazole-9-ethoxy-carbonylhydrazine and its application for determination of aldehydes from alcoholic beverage using high-performance liquid chromatography with fluorescence detection and enhance mass spectrometric identification

A pre-column derivatization method for the sensitive determination of aldehydes using the tagging reagent 1,2-benzo-3,4-dihydrocarbazole-9-ethoxy-carbonylhydazine (BCEC) followed by high-performance liquid chromatography with fluorescence detection and enhance mass spectrometric identification has been developed. The chromophore of fluoren-9-methoxy-carbonylhydrazine (Fmoc-hydrazine) reagent was replaced by 1,2-benzo-3,4-dihydrocarbazole functional group, which resulted in a sensitive fluorescence derivatizing agent BCEC. BCEC can easily and quickly label aldehydes. The maximum excitation (333 nm) and emission (390 nm) wavelengths were essential no change for all the aldehyde derivatives. The fluorescence intensity was substantially affected by the solvents, being higher in organic than protic solvents. Derivatives are sufficiently stable to be efficiently analyzed by high-performance liquid chromatography. The derivatives showed an intense protonated molecular ion corresponding m/z [M + (CH₂)_n − 1]⁺ (M: molecular mass of BCEC, n: corresponding aldehyde carbon atom numbers) under positive-ion mode. The collision-induced dissociation of protonated molecular ion formed products at m/z = 245.7.0, m/z = 263.7 and m/z = 217.7, and corresponding the cleavage of CH₂OCO, CH₂OCO and NCH₂CH₂ bonds, respectively. Studies on derivatization demonstrated excellent derivative yields in the presence of trichloroacetic acid (TCA) catalyst. Maximal yields close to 100% were observed with a 10- to 15-fold molar reagent excess. Separation of the derivatized aldehydes has been optimized on ZORBAX Eclipse XDB-C₈ column with aqueous acetonitrile in conjunction with a binary gradient elution. Excellent linear responses were observed at the concentration range of 0.08-16.65 μmol/L with coefficients of >0.9999. Estimated detection limits for the aldehydes, obtained by successive dilution of a derivatized standard solution containing 16.65 μmol/L of each aldehyde (at a signal-to-noise ratio = 3:1), are from 3.75 to 16.65 fmol.     

Development of a sensitive fluorescent derivatization reagent 1,2-benzo-3,4-dihydrocarbazole-9-ethyl chloroformate (BCEOC) and its application for determination of amino acids from seeds and bryophyte plants using high-performance liquid chromatography with fluorescence detection and identification with electrospray ionization mass spectrometry

A pre-column derivatization method for the sensitive determination of amino acids and peptides using the tagging reagent 1,2-benzo-3,4-dihydrocarbazole-9-ethyl chloroformate (BCEOC) followed by high-performance liquid chromatography with fluorescence detection has been developed. Identification of derivatives was carried out by liquid chromatography/electrospray ionization mass spectrometry (LC/ESI-MS/MS). The chromophore of 2-(9-carbazole)-ethyl chloroformate (CEOC) reagent was replaced by 1,2-benzo-3,4-dihydrocarbazole functional group, which resulted in a sensitive fluorescence derivatizing reagent BCEOC. BCEOC can easily and quickly label peptides and amino acids. Derivatives are stable enough to be efficiently analyzed by high-performance liquid chromatography. The derivatives showed an intense protonated molecular ion corresponding m/z (M + H)⁺ under electrospray ionization (ESI) positive-ion mode with an exception being Tyr detected at negative mode. The collision-induced dissociation of protonated molecular ion formed a product at m/z 246.2 corresponding to the cleavage of CO bond of BCEOC molecule. Studies on derivatization demonstrate excellent derivative yields over the pH 9.0-10.0. Maximal yields close to 100% are observed with a 3-4-fold molar reagent excess. Derivatives exhibit strong fluorescence and extracted derivatization solution with n-hexane/ethyl acetate (10:1, v/v) allows for the direct injection with no significant interference from the major fluorescent reagent degradation by-products, such as 1,2-benzo-3,4-dihydrocarbazole-9-ethanol (BDC-OH) (a major by-product), mono-1,2-benzo-3,4-dihydrocarbazole-9-ethyl carbonate (BCEOC-OH) and bis-(1,2-benzo-3,4-dihydrocarbazole-9-ethyl) carbonate (BCEOC)₂. In addition, the detection responses for BCEOC derivatives are compared to those obtained with previously synthesized 2-(9-carbazole)-ethyl chloroformate (CEOC) in our laboratory. The ratios AC_BCEOC/AC_CEOC = 2.05-6.51 for fluorescence responses are observed (here, AC is relative fluorescence response). Separation of the derivatized peptides and amino acids had been optimized on Hypersil BDS C₁₈ column. Detection limits were calculated from 1.0 pmol injection at a signal-to-noise ratio of 3, and were 6.3 (Lys)-177.6 (His) fmol. The mean interday accuracy ranged from 92 to 106% for fluorescence detection with mean %CV < 7.5. The mean interday precision for all standards was <10% of the expected concentration. Excellent linear responses were observed with coefficients of >0.9999. Good compositional data could be obtained from the analysis of derivatized protein hydrolysates containing as little as 50.5 ng of sample. Therefore, the facile BCEOC derivatization coupled with mass spectrometry allowed the development of a highly sensitive and specific method for the quantitative analysis of trace levels of amino acids and peptides from biological and natural environmental samples.