Isotope pattern deconvolution as a successful alternative to calibration curve for application in wastewater-based epidemiology.

An isotope pattern deconvolution (IPD) quantification method has been applied for the determination of five substances (amphetamine, benzoylecgonine, cocaine, methamphetamine and MDMA) in wastewater for the application in wastewater-based epidemiology (WBE). A previously validated method that used a calibration curve for quantification was modified to apply IPD. The two approaches were compared in terms of analytical uncertainty in recovery studies of quality control samples, i.e. six wastewater samples from different geographical origins spiked at two concentration levels. Both methods were reliable as they passed (z-score < 2) in an interlaboratory exercise. After 60 individual determinations, IPD provided 11 results outside recovery limits (70-120%) while the previous method produced 31 adverse results. All mean values for IPD were accurate whereas 6 out of 10 results showed RSD values higher than 30% or recoveries outside limits when using the former method. Moreover, the calculated method bias for the latter doubles that of IPD, which, in turn, makes the combined uncertainty (u(c)) much higher. Consequently, a simple change of data treatment-IPD quantification methodology-resulted in a lower uncertainty of the estimated illicit drug concentration, one of the main steps contributing to the final uncertainty in the normalized daily drug consumption through WBE. The current study demonstrated that the employment of IPD can also be very interesting for future applications of WBE, especially when matrix effects are high, complicating accurate quantification. In addition, when a high number of samples and/or compounds need to be analysed, IPD is faster than calibration and, eventually, cost-effective when isotopically labelled internal standard is highly expensive.

Isotope dilution LC-ESI-MS/MS and low resolution selected reaction monitoring as a tool for the accurate quantification of urinary testosterone.

A new analytical method for the quantification of testosterone in human urine samples by isotope dilution mass spectrometry is proposed. A standard solution of 13C2-testosterone is added to the samples at the beginning of the sample preparation procedure and then the measurements are carried out by UHPLC-ESI-MS/MS. In the proposed method, the resolution of the first quadrupole of the tandem MS instrument is reduced to transmit the whole precursor ion cluster to the collision cell and measure the isotopic distribution of the in-cell product ions with a small number of SRM transitions. The construction of a methodological calibration graph is avoided using a labelled analogue previously characterised in terms of concentration and isotopic enrichment in combination with multiple linear regression. In this way, the molar fractions of natural and labelled testosterone are calculated in each sample injection and the amount of endogenous testosterone computed from the known amount of labelled analogue. Recovery values between 97 and 107% and precisions between 0.4 and 3.7% (as %RSD) were obtained for testosterone concentrations in urine in the range of 1 to 8 ng g-1. The proposed low resolution SRM methodology was compared for the analysis of human urine samples with the traditional IDMS method based on a calibration graph and the IDMS method based on multiple linear regression combined with standard resolution SRM. A similar accuracy and precision was obtained by the three tested approaches. However, using the low resolution SRM method there was no need to resort to calibration graphs or to specific dedicated software to calculate isotopic distributions by tandem MS and a higher sensitivity was obtained. The proposed low resolution SRM method was successfully applied to the analysis of the certified freeze-dried human urine NMIA MX005.