para-aminobenzoic acid used as a marker for completeness of 24 hour urine: assessment of control limits for a specific HPLC method

Abstract

Objective and design: The study comprised three protocols. Protocol 1 compared a HPLC method with the commonly employed colorimetric diazocoupling method. Protocol 2 examined, if the last dosage of p-aminobenzoic acid (PABA) could be advanced in the old to allow for a delayed age-dependent urinary excretion of PABA. Protocol 3 established limits for recovery of PABA in 24 h urine applying the HPLC method. Subjects and setting: A total of 151 healthy volunteers participated in the study of which 140 were accepted. In protocol 1: 37 subjects aged 20–78 y were included. All subjects took PABA as recommended (80 mg orally at 08.00, 12.00 and 18.00 h). Protocol 2: compared urinary PABA excretion in two groups of 80 y old subjects who had their last PABA dosage administered at 15.00 h (n=16) and at 18.00 h (n=31), respectively. Protocol 3: comprised 56 subjects aged 20–80 y. In the younger age group (20–59 y; n=34) PABA was taken as recommended, whereas in the older age group (60–80 y; n=22) the last PABA dosage was advanced three hours. Results: Protocol 1: HPLC gave significantly lower PABA recovery results compared to colorimetry, the difference between methods being 23.9±8.5 mg/24 h (P<0.001). Protocol 2: higher PABA recoveries were demonstrated with the advanced dosage schedule compared to the recommended schedule (208±14 mg/24 h vs 181±22 mg/24 h; P<0.001). Protocol 3: PABA recovery with HPLC was 211±12 mg/24 h, and the lower limit comprising 95% of subjects was 187 mg/24 h. Similar PABA recoveries were demonstrated in the younger subjects and the older subjects (211±11 mg/24 h vs 211±13 mg/24 h; NS). Conclusion: An advanced dosage schedule for PABA in the aged is recommended. Because of lower recoveries with HPLC, the low limit for recovered PABA in a complete 24 h urine differs from the limit based on colorimetry. This study found a limit of 187 mg/24 h corresponding to the lower 95% confidence limit for a single subject. Sponsorship: Supported by the National Food Agency, the Velux Foundation of 1981, and the Health Insurance Foundation.