MSACL 2024 Abstract

1. Problem
While preparing a set of four calibrators for six arsenic species in blank human pooled urine, one of the arsenic species (arsenobetaine, AsB) had poor recoveries (0~30%) in calibrators 1-2 along with the presence of an unexpected arsenic peak. Another arsenic species (arsenocholine, AsC) had lower than expected recoveries in calibrator 1-2 (~72%). Only calibrator 4 had acceptable recoveries (within 10% of target) for AsB and AsC. The remaining arsenic species included dimethylarsonic acid (DMA), monomethylarsonic acid (MMA), AsV and AsIII and had an average recovery of 93 ± 3% to target across the four calibrators.

2. Method Information
•50μL urine diluted with 950μL diluent + internal standard (Sb) solution
•Agilent 1260 Infinity II LC
•Agilent 7700 ICP-MS
•PRP-X100 Anion Exchange HPLC column (150x4.6mm), with KrudKatcher Classic HPLC in-Line filter
•MPA – 20mM Ammonium Carbonate + 3% Methanol, pH 8.7
•MPB – 50mM Ammonium Carbonate + 3% Methanol, pH 8.0
•12min analytical step gradient, Flow 1mL/min
•Column oven 20°C
•50μL injection volume

3. Troubleshooting Steps
Three additional lots of blank human pooled urine were spiked with AsB and AsC at a calibrator 1 level (5μg/L) and left on the benchtop overnight. Two of the lots had AsB and AsC recoveries within ±10% of target while the third lot had significant degradation of AsB (<10% recovery) and AsC (80% recovery) along with the presence of the unknown arsenic peak. After a literature search, it has been reported that microorganisms are capable of AsB biotransformation. The four blank pooled urine lots were sent to be cultured and the two lots with stable AsB and AsC recoveries had no growth. The other two lots with AsB and AsC degradation had organisms present and were identified by MALDI-TOF MS as Pseudomonas fluorescens, Serratia species, Proteus vulgaris, and Achromobacter species. These organisms were isolated, inoculated into sterile urine spiked with AsB and AsC each at 5μg/L, and left overnight on the benchtop. Degradation of AsB and AsC (<20% recoveries) along with the appearance of the unknown arsenic species were observed in the Pseudomonas fluorescens and Achromobacter species inoculated urine. When urine was inoculated with Serratia species, there was low AsC recovery (~34%) while the AsB species had enhanced recovery (164%). The Proteus vulgaris and sterile urine (control) had AsB and AsC recoveries within ±10% of target.
4. Outcome
The calibrators were prepared in synthetic urine instead of human pooled urine to avoid preparing calibrators in urine contaminated with bacteria.