Toward single-calibrant quantification in HPLC. A comparison of three detection strategies: Evaporative light scattering, chemiluminescent nitrogen, and proton NMR

被引:79
作者
Lane, S [1 ]
Boughtflower, B [1 ]
Mutton, I [1 ]
Paterson, C [1 ]
Farrant, D [1 ]
Taylor, N [1 ]
Blaxill, Z [1 ]
Carmody, C [1 ]
Borman, P [1 ]
机构
[1] GlaxoSmithKline, CASS, Stevenage SG1 2NY, Herts, England
关键词
D O I
10.1021/ac050257l
中图分类号
O65 [分析化学];
学科分类号
070302 ; 081704 ;
摘要
There is an urgent need for detection technologies that enable accurate and precise quantification of solutions containing small organic molecules in a manner that is rapid, cheap, non-labor-intensive, readily automated, and without a requirement for specific analyte standards. We provide a theoretical analysis that predicts that the logarithmic nature of the working domain of the evaporative light-scattering detector (ELSD) will normally bias toward underestimation of chromatographically resolved impurities, resulting in an overestimation of analyte purity. This analysis is confirmed by experiments with flow injection analysis (FIA) and gradient reversed-phase high performance liquid chromatography (RP-HPLC). Quantification is further compromised by the dependence of response parameters on the matrix composition and hence on the retention time of the analyte. Attempts were made to ameliorate these problems by using the response surface of a single compound to calibrate throughout the HPLC gradient. A chemiluminescent nitrogen detector (CLND) was also used in a similar manner, and the performance of the two techniques were compared against those of each other and that of a reference standard technique. A protocol for this purpose was developed using proton nuclear magnetic resonance (H-1 NMR) and the ERETIC method to enable quantification by integrating proton signals. The double-blind comparison exercise confirmed molar nitrogen CLND response to be sufficiently stable and robust across a methanol gradient to be used with a single external nitrogenous calibrant to quantify nitrogen-containing compounds of known molecular formula. The performance of HPLC-CLND was very similar to that of NMR, while that of HPLC-ELSD was seen to be significantly worse, showing it to be unsuitable for the purpose of single-calibrant quantification. We report details and experience of our use of RP-HPLC-CLND-MS to characterize and quantify small amounts of solutions of novel compounds at nominal levels of 10mM in microtiter plate (MTP) format.
引用
收藏
页码:4354 / 4365
页数:12
相关论文
共 40 条
[1]  
Allgeier MC, 2003, LC GC N AM, V21, P376
[2]   A new method for absolute quantitation of MRS metabolites [J].
Barantin, L ;
LePape, A ;
Akoka, S .
MAGNETIC RESONANCE IN MEDICINE, 1997, 38 (02) :179-182
[3]   UNIVERSAL DETECTION AND QUANTITATION OF SURFACTANTS BY HIGH-PERFORMANCE LIQUID-CHROMATOGRAPHY BY MEANS OF THE EVAPORATIVE LIGHT-SCATTERING DETECTOR [J].
BEAR, GR .
JOURNAL OF CHROMATOGRAPHY, 1988, 459 :91-107
[4]   High-throughput mass spectrometry for compound characterization in drug discovery [J].
Brönstrup, M .
MODERN MASS SPECTROMETRY, 2003, 225 :283-302
[5]   Evaporative light-scattering detection in the quantitative analysis of semivolatile polycyclic aromatic compounds by high-performance liquid chromatography [J].
Cebolla, VL ;
Membrado, L ;
Vela, J ;
Ferrando, AC .
JOURNAL OF CHROMATOGRAPHIC SCIENCE, 1997, 35 (04) :141-150
[6]   EVAPORATIVE ANALYZER AS A MASS DETECTOR FOR LIQUID-CHROMATOGRAPHY [J].
CHARLESWORTH, JM .
ANALYTICAL CHEMISTRY, 1978, 50 (11) :1414-1420
[7]  
CHAVESDASNEVES HJ, 1997, J HIGH RES CHROMATOG, V20, P115
[8]   Optimization of chemiluminescent nitrogen detection for packed column supercritical fluid chromatography with methanol-modified CO2 [J].
Combs, MT ;
AshrafKhorassani, M ;
Taylor, LT ;
Fujinari, EM .
ANALYTICAL CHEMISTRY, 1997, 69 (15) :3044-3048
[9]   Liquid chromatography-mass spectrometry with chemiluminescent nitrogen detection for on-line quantitative analysis of compound collections: advantages and limitations [J].
Corens, D ;
Carpentier, M ;
Schroven, M ;
Meerpoel, L .
JOURNAL OF CHROMATOGRAPHY A, 2004, 1056 (1-2) :67-75
[10]   High-throughput analysis of natural product compound libraries by parallel LC-MS evaporative light scattering detection [J].
Cremin, PA ;
Zeng, L .
ANALYTICAL CHEMISTRY, 2002, 74 (21) :5492-5500