ZAHORRA ARTIFICIAL

A robust and accurate analytical method for the quantitative determination of β-endosulfan and endosulfan sulfate in human serum was developed and validated using LC-MS/MS with electrospray ionization and MRM scanning in the negative mode.

During the validation, intra- and inter-batch accuracy and precision were demonstrated by analyzing calibration standards (STDs) and quality control samples (QCs) in three consecutive validation batches each containing the calibration standards (STD J – STD B) in duplicate to yield one calibration curve and six replicates of quality control samples (QC H – QC A). The method was shown to be specific and selective.

The selected regression models adequately described the concentration-response relationships for each compound. Based on a range that consisted of nine calibration levels, a Wagner regression equation provided the best fit for β-endosulfan, while a linear equation, weighted

by1/concentration2 provided the best fit for endosulfan sulfate. The regression models selected

during the validation were used for the quantification of the study samples.

No suitable internal standards could be found for either of the compounds. Stable isotope-labeled congeners of the compounds were not commercially available and would not have been sufficiently specific to separate their m/z values from that of the complex isotope patterns of the compounds that each contains six chlorine atoms. Furthermore, related chlorinated pesticide molecules such as dichlorodiphenyltrichloroethane (DDT) and dieldrin are not ionizable with electrospray and could therefore not been used as internal standards. These compounds were used as internal standards in published studies based on GC-MS/MS in which electron ionization was applied [20] [26]. The fact that good retention times of both compounds were achieved during chromatography and that no significant effect from normal matrix components could be evidenced, contributed to the satisfactory performance of the method without using internal standards.

Although the matrix effect assessment indicated no significant influence on the analysis by the normal matrix components, it should be noted that lipemia in the serum had noticeable effects on the analysis of both compounds. This can be ascribed to the fact that high concentrations of phospholipids in lipemic serum can possibly influence the successful formation of cations of both analytes during the negative ionization process [70].

During the first validation batch the QC at the highest concentration level (QC H) were diluted (1:1) with blank normal serum to evaluate dilution integrity and the results demonstrated that samples with concentration levels above the upper limit of quantification (ULOQ) (up to 323 ng/ml

and 48 ng/ml for β-endosulfan and endosulfan sulfate, respectively) can be accurately quantified with this method.

System suitability tests were performed during each validation batch to ensure that the instrument did not lose response (sensitivity) during the validation batches. This was also performed for the three production batches.

The lower limit of quantification (LLOQ) was confirmed and successfully quantified with a signal to noise ratio of 8.9 and 22.6 for β-endosulfan and endosulfan sulfate, respectively. No carry-over was observed as assessed during the analysis of the three validation batches. Reinjection reproducibility was demonstrated by reinjecting the second validation batch. Stability assessments were performed during the validation and as summarized in section 2.8, no indication of instability of either of the two compounds could be found during any of the assessments.

The validation results indicated that the performance of the analytical method met the acceptance criteria as stipulated in the European Medical Agency (EMA) and Food and Drug Administration (FDA) guidelines [42] [43]. The method is therefore regarded to be suitable for the quantification of β-endosulfan and endosulfan sulfate in human serum samples over a concentration range of 0.800 ng/ml to 200 ng/ml and 0.117 ng/ml to 30 ng/ml, respectively.

The applicable clinical protocol (REC REF: 279/2005) mentions the detection of endosulfan and other pesticides in surface and groundwater of rural Western Cape areas [8] [9] [10]. The samples analyzed in this study originated from farm workers active in the same area and therefore thought to have been exposed to the pesticides. However, no evidence for the exposure to endosulfan pesticides could be found in any of the serum samples. This was notwithstanding the fact that the STDs and QCs in the production batches conformed to the same standards found during the validation performance. The fact that none of the compounds could be observed could therefore not be ascribed to poor performance of the analytical method. Furthermore, the signal to noise ratio of the LLOQs for both compounds, especially for endosulfan sulfate, was high enough to still observe sub-LLOQ concentrations of the compounds. No such observations were made. The reasons for the negative results may reside in seasonal variation in the application of pesticides used for pest control. This possibility can be explored by correlating the pesticide products used during or before the period of sampling. However, it may indicate that the time of sampling was too long after exposure or exposure was not high enough to result in levels of the compounds within the calibration range.

CHAPTER 3

DEVELOPMENT AND VALIDATION OF A BIOANALYTICAL

METHOD FOR THE DETERMINATION OF DIALKYLPHOSPHATE

METABOLITES IN HUMAN URINE BY LC-MS/MS

3.1 INTRODUCTION

As indicated in the rationale for the clinical trial (REC REF: 279/2005), South Africa is the highest pesticide user in Southern Africa and exposure to these harmful pesticides needs to be determined to incorporate protective practices. Pesticide exposure can occur through a number of sources as indicated in Chapter 1 and the approach to biological monitoring for organophosphates is based on the analysis of diakylphosphate metabolites in urine [71] [72] [73]. Organophosphates are rapidly hydrolyzed to the diakylphosphate (DAP) metabolites detectable in urine, and can be measured several days after exposure [74]. Identification of these metabolites can be used to monitor the occasional exposure to organophosphate pesticides [75] and the non-invasive sampling procedure is preferable [76].

Previous investigations have shown that the use of first morning void urine samples accurately represent total daily exposure to organophosphates [77] although variable urine production may influence the concentration levels [71]. The ubiquitous use of house-hold pesticides containing organophosphates poses a further disadvantage when measuring the DAP metabolites as markers for specific agricultural exposure [71]. Environmental routes of exposure to pesticides through contaminated food, soil, water and spray drift in addition to occupational exposure might be important for rural residents in the Western Cape. The use of a control group of subjects to act as a “population background level” is therefore essential.

Due to the superior specificity and sensitivity that can be achieved with high performance liquid chromatography with tandem mass spectrometer detection (LC-MS/MS), this technique was applied in this study to quantitatively measure three of the DAP metabolites, dimethyl phosphate (DMP), diethyl phosphate (DEP), and dimethyl thiophosphate (DMTP), using the corresponding deuterated molecules as internal standards. One method was developed to extract the three metabolites from urine and to determine the concentrations by LC-MS/MS. Although other known metabolites could also be assessed, the lack of appropriate internal standards to accurately quantify all of the molecules restricted the method to the three mentioned above.

The developed and partially validated method used for the quantitative determination of dialkylphosphate (DAP) metabolites in urine was performed at the University of Cape Town. This

method was used to investigate the possible correlation between exposure to the pesticides and urinary levels of the metabolites, data which can also be compared to literature reports.

3.2 CHEMICALS AND MATERIALS USED