Ultra-trace element analysis of human follicular fluid by ICP-MS/MS: pre-analytical challenges, contamination control, and matrix effects

Abstract

Follicular fluid (FF), which is the fluid that envelops the developing oocyte (egg cell) in the ovary, can be analyzed to assess trace element content as well as to determine potential exposure to toxic elements in women seeking in vitro fertilization (IVF) treatment. Such measurements may be useful in establishing associations with potential adverse effects on oocyte viability and subsequent pregnancy outcomes. The principal goal of this study was to leverage the next generation of inorganic mass spectrometry based on ICP-MS/MS to address the numerous analytical challenges of (ultra-)trace element analysis of human FF specimens. Ultra-trace element measurements are defined by the Clinical Laboratory Standards Institute as fluid concentrations below 10 μg L^-1 or tissue mass fractions below 1 μg g^-1. Stringent pre-analytical procedures were developed to minimize exogenous contamination during FF specimen collection and storage in a prospective study of 56 women seeking IVF treatment. ICP-MS/MS instrumental parameters were carefully optimized, and the method validated for 11 biologically important elements that included 4 at trace levels (Cu, Se, Sr, and Zn) and 7 at ultra-trace levels (As, Cd, Co, Mo, Mn, Hg, and Pb). Method limits of detection (LODs) for ultra-trace elements varied from 5.6 ng L^-1 for Cd to 0.11 μg L^-1 for Mo. A total of 197 human FF specimens were analyzed using the proposed ICP-MS/MS method with 84% of specimens detectable for Pb and 100% detectable for Co, Cu, Mn, Mo, Sr, and Zn. The method based on ICP-MS/MS was compared to a previous method developed for FF using SF-ICP-MS.

Keywords: ICP-MS/MS; QQQ-ICP-MS; contamination; human follicular fluid; trace elements; ultra-trace elements.

Figure 1

ICP-MS/MS signal (cps) for Cd and Pb in untreated (filled circles) and acid-washed (open circles) autosampler tubes from four manufactures: Nalgene (black), Grenier (red), Sarstedt (blue), and Falcon (green). For each group, the individual data points are shown along with the mean ± 95% CI.

Figure 2

Concentrations of Cd and Pb are plotted in 1) acid-washed Sarstedt autosampler vials (AW-Sarstedt); 2) untreated VWR tubes (UT-VWR); and 3) acid-washed VWR tubes (AW-VWR). For each group, the individual data points (filled circles) are shown along with the mean ± 95% CI.

Figure 3

Within-run signal stability for As in a base serum sample injected 100 times (~22 hours) using three nebulizers: MicoMist (purple), MiraMist (blue), and SeaSpray (black). The stability is expressed in panels A, B, and C as As concentration, instrument signal (cps), and the ratio of As:Rh (analyte cps to internal standard cps).

Figure 4

Comparison of values obtained for two human FF-based QC materials for 1) material characterization using standard additions as a reference method (filled in circles) and 2) analyses against an external matrix-matched calibration as part of the method validation and sample batches (open circles). The error bars represent the expanded uncertainty, U, calculated as $3\sqrt{{\left(\frac{SD}{n}\right)}^2}.$

Figure 5

The difference between values obtained by ICP-MS/MS (QQQ) and SF-ICP-MS is shown as a function of the mean concentration of As, Cd, Co, Hg, Mn, Mo, and Pb in 60 common samples. For these elements a large proportion of the data were below the method LOQ for either the ICP-MS/MS method or the SF-ICP-MS method. Thus, acceptability criteria (black dashed lines) are proposed as ± an absolute criterion (shown per element) or ±15%, whichever is greater. Method LOQs are shown for the QQQ method (red dashed line) and SF method (solid red line).

Figure 6

Values obtained by ICP-MS/MS are shown as a function of those reported by SF-ICP-MS for Cu, Se, Sr, and Zn in 60 common samples. The solid purple line dashed black lines represent y=x ±15%.

Figure 7

Values obtained by ICP-MS/MS for As (black), Cd (red), Hg (blue), and Pb (green) in the 197 FF samples measured as part of the present study (filled in circles) compared to those measured in 60 of the FF samples that were originally measured by SF-ICP-MS (open circles). The geometric mean ± geometric standard deviation are shown for each group.