Enhanced chromatography using multiphoton detection

#This application is entitled to the priority of provisional patent application Ser. No. 60/001,129, filed Jul. 13, 1995, and is related to commonly owned U.S. Pat. No. 5,532,122, issued Jul. 2, 1996, and to a commonly owned application filed Jun. 25, 1996 entitled "Ultralow Background Multiple Photon Detector," the disclosures of all of which are incorporated herein by reference.

#1. Field of the Invention

#The invention provides chromatographic methods and reagents suitable for the detection and quantification of analytes of interest at femtomolar (10.sup.-15 M) concentrations. More specifically, the invention relates to binding a radioactive derivatizing agent to an analyte of interest present in a mixture at low concentration, separating the analyte of interest, and detecting it using multiphoton detection.

#The invention is better understood by reading the following detailed description with reference to the accompanying figures.

#FIG. 1: HPLC/multiphoton detection measurements of binary dilutions of a mixture of estradiol and testosterone.

#FIG. 2: Reproducibility of HPLC/multiphoton detection measurements of about 20 attomole/sample of the mixture of estradiol and testosterone; the integrated count rate in each of the two peaks is plotted. The data are normalized to the average of the integrated activity in each peak.

#In describing preferred embodiments of the present invention, specific terminology is employed for the sake of clarity. However, the invention is not intended to be limited to the specific terminology so selected, and it is to be understood that each specific element includes all technical equivalents which operate in a similar manner to accomplish a similar purpose.

#Multiphoton detection enhanced chromatography provides sensitivity of a few femtograms per/ml, or femtomolar concentrations. With sample sizes of 10 microliters with molecular mass of a few hundred daltons, sub-attomole sensitivity is achieved.

#Multiphoton detection as described in commonly owned U.S. Pat. No. 5,532,122 has achieved sub-zeptomole/ml (<10.sup.-21 mole/ml) sensitivity. The multiphoton detection technique applied to HPLC and TLC permits a factor of a thousand more sensitive measurements than the most popular conventional detection methods--UV absorption detection (UVD) and electrochemical detection (ECD). Techniques compatible with the use of multiphoton detection in chromatography permit significant improvement of the detection limits using Inexpensive instrumentation with sensitivity of better than 10.sup.-15 g/ml. Thus, for substances with molar masses in the few hundred dalton range, sub-attomole/ml sensitivity is achieved.