SAMPLE PREPARATION AND HIGH-PERFORMANCE LIQUID-CHROMATOGRAPHIC ANALYSIS OF DEOXYRIBONUCLEOSIDE TRIPHOSPHATES IN INDIVIDUAL RAT EMBRYOS

A rapid, robust, and sensitive method has been developed to measure co ncentrations of deoxyribonucleoside triphosphates in individual, day 1 4 rat embryos by modifying and optimizing existing methods for cellula r extracts. Significant changes include: (i) oxidative degradation of ribonucleoside triphosphates using methylamine at lower pH (decreased from 6.5 to 4.0) to improve poor HPLC peak shape of early eluting nucl eotides; (ii) glass fiber disc solid-phase extraction of the reaction mixture, which dramatically reduces impurities that interfere with nuc leotide measurement, eliminates the necessity of column regeneration, and allows mobile phase recycling; and (iii) lower ionic strength (red uced from 0.4 to 0.26 or 0.12 M ammonium phosphate) and higher pH (inc reased from 3.25 to 5.55 or 6.98, respectively) mobile phase, conditio ns which are less destructive to the column's bonded phase and silica support, thereby contributing to longer column life. Enhancements incl ude: (i) filtration of the sample prior to HPLC injection and addition of an in-line filter, guard column, and saturating precolumn of silic a in the mobile phase how, which aids substantially in extending colum n life and improves chromatographic stability, and (ii) inclusion of a n internal standard to correct for mechanical losses, Limits of determ ination at a signal to noise ratio of 6:1 range from 5.5 to 12 pmol on -column or 0.41 to 0.87 pmol/mg of embryonic tissue depending on the s pecific nucleotide. Recoveries are quantitative for all nucleotides, a nd interassay variabilities are between 5 and 7% when quantified by pe ak height. The method has also been applied successfully to analysis o f murine erythroleukemic cell cultures and this, when coupled with the embryo results, suggests its general utility.