HPLC/MPD - Pilot studies Excelllent sensitivity and reproducibility of HPLC/MPD was demonstrated for steroids and insulin. HPLC/MPD achieved 0.1 and 1.0 attommole/sample sensitivity, for steroids and insulin, respectively. We expect we will be able to achieve sub-attomole/sample sensitivity for neurotransmitters and other small molecules. A sensitivity of 10 attommole/sample has been achieved for anti-dopamine using precolumn derivatization methods.
1: Neutrotransmiter ¹²⁵I-SCH-23982 (an atagonist of dopamine) was analyzed by reverse-phase HPLC. The limits of detection were 500-fold better than when a UV detector was used. The integrated activity in the peak corresponding to the radiolabeled product changes linearly with the reagent concentration over three orders of magnitude,i.e., from a few fg/ml to a few pg/ml. Good reproducibility of HPLC/MPD was demonstrated.
Derivatization of GABA (4-aminobutanonic acid) with radiophote has been achieved followed by HPLC separation and MPD detection. The radioactive derivatizing agent (pipsyl chloride) was synthesized from p-aminobenzenesulfonic acid via diazonium salt formation and a subsequent reaction with sodium(¹²⁵I) iodine. The product was reacted in the presence of triethyamine with GABA (4-aminobutanoic acid) in benzene. Aliquots (10mL and 2mL) of the reaction mixture were injected to the HPLC system under optimized conditions. Quantation using MPD confirmed the validity of the derivatization procedure. The presence of GABA has been detected at 10 attomoles/sample level and S/B >3 has been achieved.
2: Radioiodinated sex steroids were studied, namely testosterone and estradiol. The separation was performed at room temperature using a Perkin-Elmer HPLC instrument with Cartridge C-18 Column. The solvent system consisted of acetonitrile and water. The flow rate was kept constant at 1 ml/min and 1 ml fractions were collected for 35 minutes and measured by MPD. Our experiments demonstrated that even for very diluted steroid mixture, down to 0.1 attomole/sample, HPLC/MPD resolves peaks very well (see Figure 1) , each showing a high signal-to-noise ratio. Figure 2 shows the good linearity for dilutions down to 0.1 attommole/sample. Even for this ultralow concentration, which was undetectable using prior at system, the signal can be easily distinguished from background. Figure 3 shows excellent reproducibility of HPLC/MPD for the 10 attomole/sample level. We also performed HPLC/MPD of insulin spiked in blood and urine; excellent chromatograms were obtained down to the attomole/sample level.


Figure 1. The HPLC/MPD of a mixture of estradiol and testosterone; note very well resolved peaks for 9 and 45 attomole/sample respectively