Validated method for the determination of perfluorinated compounds in placental tissue samples based on a simple extraction procedure followed by ultra-high performance liquid chromatography tandem mass spectrometry analysis

1 Validated method for the determination of perfluorinated compounds in placental tissue samples based on a simple extraction procedure prior to ultra–high performance liquid chromatography–tandem mass spectrometry analysis J. Martína, R. Rodríguez–Gómezb, A. Zafra–Gómez*b, E. Alonsoa, J.L. Vílchezb, A. Navalónb 1 Department of Analytical Chemistry, Superior Polytechnic School, University of Seville, C/ Virgen de África 7, E-41011 Seville, Spain 2 Research Group of Analytical Chemistry and Life Sciences, Department of Analytical Chemistry, University of Granada, Campus of Fuentenueva, E-18071 Granada, Spain ABSTRACT Exposure to xenobiotics during pregnancy is inevitable. Determination of perfluorinated compounds (PFCs), renowned environmental contaminants by Public Health Authorities due to its persistence, bioaccumulative effect and toxicity, is a challenge. In the present work, a method based on a simplified sample treatment involving steps of freezedrying, solvent extraction and dispersive clean-up of the extracts with C18 prior to ultra-high performance liquid chromatography–tandem mass spectrometry (UHPLC–MS/MS) analysis for the determination of 5 perfluorinated carboxylic acids (from C4 to C8) and perfluorooctane sulfonate (PFOS) in placental tissue samples is developed and validated. The most influential parameters affecting the extraction method and the clean-up step were optimized using design of experiments. The method was validated using matrix-matched calibration. Found detection limits (LOD) ranged from 0.03 to 2 ng g-1 and quantification limits (LOQ) from 0.08 to 6 ng g-1, while inter- and intra-day variability was under 14% in all cases. Recovery rates for spiked samples ranged from 94 to 113%. The method was satisfactorily applied for the determination of compounds in human placental tissue samples collected at the moment of delivery from 25 randomly selected women. Keywords: Perfluorinated compounds; Placental tissue; Dispersive solid phase extraction; UHPLC–MS/MS _________________ * Corresponding author 2 E–mail address: [email protected] (A. Zafra-Gómez) 3 1. Introduction Exposure to xenobiotics during pregnancy is inevitable. They are likely to act not only directly on the fetus but also on the placenta itself which may affect its ability to support pregnancy [1]. Endocrine disruptors (EDs) are ubiquitous in the environment around us and as a consequence in human bodies. There is a growing concern about the potential health effects of exposure to various environmental chemicals during pregnancy and infancy. Mono-2- ethylhexyl phthalate (MEHP), octylphenol (OP), 4-nonylphenol (4-NP) or polybrominated diphenyl ethers (PBDEs) have been detected in physiologically relevant compartments within pregnant women and the developing fetuses, such as maternal urine, cord blood, breast milk, meconium, placenta and amniotic fluid in several studies [2-8], showing that pregnant women and their fetuses are exposed to those chemicals. Perfluorinated compounds (PFCs) have been recognized as an important class of potential contaminants by Public Health Authorities due to its persistence, bioaccumulative effect and toxicity [9]. Since their first production in 1947, PFCs have been used in a wide range of commercial and industrial applications such as polymers, metal plating and cleaning, surfactants, lubricants, pesticides, coating formulations, inks, varnishes, firefighting foam, and stain/water repellents for leather, paper and textiles. Worldwide human exposure to PFCs has been confirmed [10-13]. For the general population the major source of contamination arises from food and sometimes drinking water [12, 14-16]. Human exposure also arises from indoor and ambient air and house dust. Perfluorooctane sulfonate (PFOS) is generally the most abundant PFCs found in humans, the second usually being perfluorooctanoic acid (PFOA) [9]. An advanced estimation (2012) of dietary exposure was ~5–10 ng/kg bw/day for PFOS, 4–7 ng/kg bw/day for PFOA, the most important contributors being fish and seafood, fruit and fruit products [17]. Children, especially toddlers, are more exposed than adults (2-3 times higher) on a body weight basis, due to higher relative food consumption and to hand-to- mouth transfer from impregnated carpets and ingestion of dust, resulting in higher PFCs serum levels than for adults [14, 18, 19]. In utero and postnatal exposures are of particular concern. PFCs cross the placental barrier, exposing neonates via their mother's blood [20-22]. In addition, first scientific evidences suggesting that PFCs would be responsible for reproductive disorders in humans have been recently published [23-29]. Butenhoff et al. [26] published a work on the role of PFCs in the onset of health troubles affecting the reproductive function when exposure occurs during critical stages of the development, i.e. the perinatal 4 period. Fei et al. [25] observed an association between high serum levels of PFOS and PFOA and a longer time to pregnancy. Effects of in utero exposure to PFCS on the female reproductive functions have also been reported. In a recent study, carried out by Kristensen and co-workers [29], daughters who were exposed in utero to levels of PFOA that were higher than the reference group showed a later age of menarche. In particular, a number of animal toxicology studies have shown that exposure to PFOS and PFOA can alter ovarian function [27] and affect the development of mammary gland tissue [28]. In this context, the characterization of the PFCs transfer, if any, from the mother to the fetus (through placenta) and/or to the newborn (through breastfeeding) is acutely expected. Biomonitoring data reflecting the internal PFCs exposure levels in the general population have already been provided in breast milk, maternal and cord serum from different states/countries [30]. However, there is limited data on the in utero exposure levels of newborns to these PFCs so far. Most of the researches on placental transfer of PFCs were conducted by animal or in vitro experiments instead of human studies. Therefore, the validation of analytical methods to determine PFCs in human placental tissue is of special scientific interest. The preferred technique for the analysis of these compounds has been high-performance liquid chromatography-tandem mass spectrometry in negative electrospray ionization mode (LC/ESI–MS/MS) [31-32]. The isolation of analytes from this complex biological matrix is a critical aspect even when a chromatographic technique is employed due to selectivity and sensitivity issues. Some techniques such as microwave-assisted extraction (MAE) or pressurized liquid extraction (PLE) could be good alternatives to maximize the extraction efficiency, but the application of these techniques involves drastic conditions, specific technical resources and not very clean final extracts including ion suppression, which is critical when mass spectrometry is employed as detection technique [33]. Solvent extraction with vortex homogenization followed by a clean-up of the extract based on dispersive solidphase extraction (d-SPE) was chosen in our study as extraction technique because its simplicity, low cost, short extraction time, low volume of solvents required and its widespread use in most of the laboratories focused on routine analyses. To our knowledge, there is a lack of published literature on PFCs determination in placental tissue. The aim of the present work was to validate a fast, simple, accurate and sensitive UHPLC–MS/MS method for the determination of PFCs in freeze-dried human placental tissue samples. After validation, the method was satisfactorily applied to determine the free content of target compounds in samples collected from 25 unknown volunteers. 5 2. Experimental 2.1. Chemicals and reagents All reagents were analytical grade unless otherwise specified. Water (18.2 MΩ cm) was purified using a Milli-Q system from Millipore (Bedford, MA, USA). Perfluorobutanoic acid (PFBuA) (98%), perfluoropentanoic acid (PFPeA) (97%), perfluorohexanoic acid (PFHxA) (≥ 97%), perfluroroheptanoic acid (PFHpA) (99%), perflurorooctanoic acid (PFOA) (96%), and perfluorooctanesulfonic acid (PFOS) (≥ 98%) and perfluoro-n-[1,2,3,4-13C4]octanoic acid (MPFOA) were purchased from Sigma-Aldrich (Madrid, Spain). Stock solutions of each compound, at a concentration of 1000 mg L-1, were prepared in methanol and stored at -20 ºC. Working solutions were prepared by diluting the stock standard solutions in methanol or in the initial mobile phase immediately before use. These solutions were stored at 4 ºC and prepared fresh weekly. All solutions were stored in dark glass bottles to prevent photodegradation. Anhydrous magnesium sulfate was provided by Panreac (Barcelona, Spain). PSA sorbent (primary secondary amine, 40–60 µm) was purchased from Scharlab (Barcelona, Spain) and BAKERBONDs octadecyl C18 sorbent (40 µm particle size) was provided by J. T. Baker (Deventer, The Netherlands). LC–MS grade methanol, water and acetonitrile, acetic acid and ammonia (25%, w/v) were purchased from Sigma-Aldrich. 2.2. Instrumentation and software UHPLC–MS/MS analysis was performed using a Waters Acquity UPLCTM H-Class (Waters, Manchester, UK), consisting of Acquity UPLCTM binary solvent manager and Acquity UPLCTM sample manager. A Xevo TQS tandem quadrupole mass spectrometer (Waters) equipped with an orthogonal Z-sprayTM electrospray ionization (ESI) source was used for PFCs detection. An Acquity UPLC® BEH C18 (50 mm × 2.1 mm i.d., 1.7 µm particle size) and a CORTECS UPLCTM C18 (Waters) column (50 mm × 2.1 mm; 1.6 µm particle size) from Waters (UK) were assayed as chromatographic columns. Placental tissue samples were freeze-dried using a SCANVAC CoolSafe™ freezedryer (Lynge, Denmark). A Branson digital sonifier (Danbury, CT, USA), a vortex-mixer (IKA, Staufen, Germany), an ultrasound- HD bath (Selecta, Barcelona, Spain), a Spectrafuge™ 24D centrifuge from Labnet 6 International, Inc. (New Jersey, USA) and a sample concentrator (Stuart, Staffordshire, UK) were also used. Statgraphics Plus software version 5.1 (Statpoint Technologies Inc., Virginia, USA) was used for statistical treatment of data. 2.3. Sample collection and storage Placenta tissue samples were obtained from health women living in Granada, Spain. Samples were anonymized, frozen at -20 ºC and stored until analysis in our laboratory. The study was performed in compliance with the Ethical Principles for Medical Research Involving Human Subjects issued by the World Medical Association, and all volunteers signed the informed consent form. Human placental tissue samples were collected from volunteers at the moment of delivery. In order to ensure the homogeneity and representativeness of the whole placenta tissue, each one was accurately examined, weighed and fragmented. Then, half of the placenta (including maternal and fetal sides and central and peripheral parts) was placed in the glass container of a mixer for its homogenization. Once homogenized, aliquots of 25 g were stored frozen at –86 ºC within 90 min of its collection. Samples were anonymized to preserve the confidentiality of patients. All volunteers signed their informed consent to participate in the study. Before processing, placental tissue aliquots were additionally homogenized using an ultrasonic spindle. The container was placed in a glass full of ice in order to avoid sample heating and the spindle was in direct contact with the placental tissue. Ultrasound setting consisted in pulses duty cycles of 30 s followed by 30 s without sonication, until complete 5 min of effective radiation. Once the samples were homogenized, they were frozen at −86 ºC and stored confidentially and anonymously until analysis. 2.4. Basic procedure 2.4.1. Preparation of spiked samples Due to the absence of certified materials for these compounds in this matrix, 6 g of spiked sample were prepared containing the analytes at seven different concentration levels, from 0.025 to 62.5 ng g-1, for calibration standards and 1.25, 6.25, 12.5 and 25 ng g-1, for quality control and validation of the method. Spiked samples were accurately stirred and slightly heated (35 ºC) to homogenize. Then, aliquots of 1 g were weighted in 8 mL glass 7 vials and fortified with 200 µL of a methanolic solution (62.5 ng mL-1) of the surrogate MPFOA (final concentration 12.5 ng g-1 in placental tissue). After shaking for 10 min, the spiked samples were ready for the experiments. 2.4.2. Sample treatment An aliquot of placental tissue sample (1 g) was weighed in an 8 mL glass vial, fortified with 200 µL of methanol containing 62.5 ng mL-1 of MPFOA, shaken for 5 min and freezedried. The sample was homogenized with 5 mL of acetonitrile in vortex for 1 min. Then, the mixture was centrifuged for 10 min at 4050 × g. In order to decrease the matrix co-extractives in the extract that could cause the matrix effect, a clean-up of the extract based on d-SPE was carried out. The extract was transferred to an 8 mL polypropylene conical tube containing 108 mg of C18 sorbent. The mixture was hand-shaken for 2 min and centrifuged for 5 min at 4050 4050 × g. The organic phase (acetonitrile) containing the analytes was separated into a glass vial and then evaporated under a nitrogen stream at room temperature and the final residue was dissolved with 250 µL of the initial mobile phase. After stirring for 30 s in vortex and centrifugation for 5 min at 16,300 × g, the sample was ready to be injected into the LC system. 2.4.3. Ultra-high performance liquid chromatography–tandem mass spectrometry conditions Chromatographic separation of compounds was performed using a CORTECS UPLCTM C18 (Waters) column (50 mm × 2.1 mm; 1.6 µm particle size). The compounds were separated using a gradient mobile phase consisting of a buffer solution acetic acid/ammonium acetate (pH 4.4) (solvent A) and methanol (solvent B). Gradient conditions were: 0.0-5.0 min, 20-100% B and back to 20% in 0.1 min and kept for 1.9 min to equilibrate the column. Flow rate was 0.3 mL min-1. The injection volume was 10 µL. The column temperature was maintained at 40 ºC. Total run time was 7 min. The tandem mass spectrometer was operated in the selected reaction monitoring (SRM) mode and Q1 and Q3 quadrupoles were set at unit mass resolution. ESI was performed in negative ion mode. The ion source temperature was maintained at 150 ºC. Instrument parameters were as follows: capillary voltage, 0.60 kV; source temperature, 150 ºC; desolvation temperature, 500 ºC; cone gas flow, 150 L h-1; desolvation gas flow, 500 L h-1; collision gas flow, 0.15 mL min-1, and nebulizer gas flow, 7.0 bar. Nitrogen (99.995%) was 8 used as cone and desolvation gas, and argon (99.999%) was used as a collision gas. Dwell time was 25 ms. Table 1 shows the parameters and the mass transitions for each compound. Table 1 2.4.4. Method validation Validation in terms of linearity, selectivity, accuracy (trueness and precision) and sensitivity, was performed according to the US Food and Drugs Administration (FDA) guideline for bioanalytical assay validation [34]. Linearity of the calibration graphs was tested using the determination coefficients (%, R2) and the P-values of the lack-of-fit test (%, Plof) [35]. The specificity of the method was determined by comparing the chromatograms of blank with the corresponding spiked placental tissue. Due to the absence of certified materials, in order to evaluate the trueness and the reproducibility of the method, a study with spiked placenta samples, at three concentrations levels (6.25, 12.5 and 25 ng g-1 for PFBuA, PFPeA and PFHxA and 1.25, 6.25 and 12.5 ng g-1 for PFHpA, PFOA and PFOS) was performed. Precision, expressed as relative standard deviation (%, RSD) was determined from triplicate spiked samples during the same day and in six different days, and the trueness was evaluated by a recovery assay. The recovery of the tested compounds in placenta samples was evaluated by comparing the known concentration in spiked samples with the concentration of each compound determined using the method proposed. Finally, LODs and LOQs were calculated by taking into consideration the standard deviation of residuals, sy/x, the slope, b, of the calibration graphs and an estimate so obtained by extrapolation of the standard deviation of the blank (Analytical Methods Committee). The LOD was 3·so and the LOQ was 10·s0. 2.4.54. Quality assurance and quality control Validity of the analytical results was verified by some simple quality assurance and quality control (QA/QC) measures. Procedural blanks were injected to monitor for background contamination. Blanks were processed in the same way as the samples and injected into the UHPLC–MS/MS system. No quantifiable amounts of target compounds were detected. On the other hand, in order to evaluate possible contaminations and the variability of the instrumental analysis, standards (spiked blank samples at 0 and 25 ng g-1) and a standard in the initial mobile phase (100 ng mL-1) were injected by triplicate every 20 samples. 9 3. Results and discussion 3.1. Liquid chromatographic-mass spectrometric analysis A BEH C18 UPLCTM column (50 mm × 2.1 mm i.d., 1.7 µm particle size) and a CORTECS UPLCTM C18 (Waters) column (50 mm × 2.1 mm; 1.6 µm particle size) were tested. Although both columns offered similar resolution for all the analytes investigated, at the retention time of the analytes eluted CORTECS UPLCTM column provided better peak shape in the shortest time with, even this last generated pressures 1000 psi lower than BEH C18 UPLCTM. Consequently, this column was the one we selected for further experiments. The effect of the mobile phase on chromatographic separation was also studied. Our aim was to obtain high sensitivity and selectivity in a short time. First, the pH of the mobile phase was studied and deionized water with different additives was studied as solvent A. Acetic acid (from 0% to 0.2%, v/v), ammonia (from 0% to 0.050%, w/v) and mixtures of them were assayed. Higher responses and better peak shapes were obtained using a mixture of 1:1 (v/v) of acetic acid (0.1%, v/v) and ammonia (0.025% w/v). MeOH, ACN and mixtures of these solvents were evaluated as organic mobile phases (solvent B). MeOH was selected because of the sensitivity, peak shapes and separation achieved. A linear gradient, as described in the previous section, was used. The injection volume was studied in order to enhance the analytical signal and consequently the LOD of the method. A range from 2.5 to 10 µL was analyzed and 10 µL was chosen as injection volume since a marked increase in sensitivity without loss of resolution was obtained. Finally, the increase of temperature from 30 to 50 ºC did not improve significantly the characteristics of chromatographic method, therefore 340 ºC was chosen as optimum. The MS/MS detection method was set up by continuous infusion of standard solutions of each individual compound (1 mg L-1) to optimize the response of the precursor ion. The mass spectrometric conditions were optimized for each compound. ESI and ESCI interfaces in positive and negative modes were evaluated. 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Vertical line shows the limit of decision to consider the significance of the factors (based on the standardized effect = estimated effect/standard error, at 95% of confidence level). Fig. 4. UHPLC–MS/MS chromatograms of: (A) a spiked blank (25 ng g-1 of each analyte); (B) a positive placental tissue sample contaminated with PFOA and PFOS (mother 24). 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 21 Figure captions Fig. 1. Influence of solvent on the extraction procedure (n = 3). Fig. 2. Influence of the extraction modality (vortex or ultrasound assisted) on the recoveries (n = 3). Fig. 3. Standardized main effect Pareto charts for the Box–Behnken design. (+) Positive effects on the response, (-) negative effects on the response. Vertical line shows the limit of decision to consider the significance of the factors (based on the standardized effect = estimated effect/standard error, at 95% of confidence level). Fig. 4. Response surface plots corresponding to the desirability function when optimizing the following pair of factors from the clean-up step with d-SPE sorbents: (A) amount of PSA vs. amount of C18; (B) amount of MgSO4 vs. amount of C18 and (C) amount of MgSO4 vs. amount of C18. Results were evaluated using a 95% confidence interval. Fig. 5. UHPLC–MS/MS chromatograms of: (A) a spiked blank (25 ng g-1 of each analyte); (B) a positive placental tissue sample contaminated with PFOA and PFOS (mother 24). Table 1 Optimized MS/MS parameters for SRM analysis. Name Abbreviation Retention time (min) Precursor Ion (m/z) SRM 1a (m/z) SRM 2b (m/z) CV (V) CE (eV) Perfluorobutanoic acid PFPuA 1.60 213 169 51.6 36 20 Perfluoropentanoic acid PFPeA 3.18 263 219 89.7 36 20 Perfluorohexanoic acid PFHxA 3.98 313 269 119 36 20 Perfluoroheptanoic acid PFHpA 4.49 363 319 333 36 20 Perfluorooctanoic acid PFOA 4.87 413 369 194 36 20 Perfluorooctane sulfonate PFOS 5.18 499 80 52 36 20 Perfluoro-n-[1,2,3,4-13C4]octanoic acid MPFOA 4.87 417 371 168 36 20 CV: Cone voltage (V); CE: Collision energy (eV); a: SRM transition used for quantification; b: SRM transition used for confirmation Table Table 2 Analytical and statistical parameters. PFBuA PFPeA PFHxA PFHpA PFOA PFOS bMP (g ng-1) 0.066 0.12 0.24 0.19 0.19 0.59 sbMP (g ng-1) 8.5·10-4 9.4·10-4 3.1·10-3 2.2·10-3 2.2·10-3 6.9·10-3 bPT (g ng-1) 0.036 0.11 0.24 0.20 0.20 0.30 sbPT (g ng-1) 1.1·10-3 6.4·10-3 4.4·10-3 7.0·10-3 7.0·10-3 1.1·10-2 tstudent 13 1 2 1 1 13 % R2 99.9 99.3 99.9 99.8 99.8 99.6 LOD (ng g-1) 1.5 2 0.5 0.03 0.03 0.03 LOQ (ng g-1) 4 6 2 0.08 0.08 0.08 LDR (ng g-1) 4.0-62.5 6.0-62.5 1.8-62.5 0.08-62.5 0.08-62.5 0.08-62.5 MP: Mobile phase; PT: Placental tissue; b: slope; sb: slope standard deviation; R2: determination coefficient; LOD: limit of detection; LOQ: limit of quantification; LDR: linear dynamic range Table Figure Click here to download high resolution image 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 21 Figure captions Fig. 1. Influence of solvent on the extraction procedure (n = 3). Fig. 2. Influence of the extraction modality (vortex or ultrasound assisted) on the recoveries (n = 3). Fig. 3. Standardized main effect Pareto charts for the Box–Behnken design. (+) Positive effects on the response, (-) negative effects on the response. Vertical line shows the limit of decision to consider the significance of the factors (based on the standardized effect = estimated effect/standard error, at 95% of confidence level). Fig. 4. Response surface plots corresponding to the desirability function when optimizing the following pair of factors from the clean-up step with d-SPE sorbents: (A) amount of PSA vs. amount of C18; (B) amount of MgSO4 vs. amount of C18 and (C) amount of MgSO4 vs. amount of C18. Results were evaluated using a 95% confidence interval. Fig. 5. UHPLC–MS/MS chromatograms of: (A) a spiked blank (25 ng g-1 of each analyte); (B) a positive placental tissue sample contaminated with PFOA and PFOS (mother 24). Table 1 Optimized MS/MS parameters for SRM analysis. Name Abbreviation Retention time (min) Precursor Ion (m/z) SRM 1a (m/z) SRM 2b (m/z) CV (V) CE (eV) Perfluorobutanoic acid PFPuA 1.60 213 169 51.6 36 20 Perfluoropentanoic acid PFPeA 3.18 263 219 89.7 36 20 Perfluorohexanoic acid PFHxA 3.98 313 269 119 36 20 Perfluoroheptanoic acid PFHpA 4.49 363 319 333 36 20 Perfluorooctanoic acid PFOA 4.87 413 369 194 36 20 Perfluorooctane sulfonate PFOS 5.18 499 80 52 36 20 Perfluoro-n-[1,2,3,4-13C4]octanoic acid MPFOA 4.87 417 371 168 36 20 CV: Cone voltage (V); CE: Collision energy (eV); a: SRM transition used for quantification; b: SRM transition used for confirmation Table Table 2 Analytical and statistical parameters. PFBuA PFPeA PFHxA PFHpA PFOA PFOS bMP (g ng-1) 0.066 0.12 0.24 0.19 0.19 0.59 sbMP (g ng-1) 8.5·10-4 9.4·10-4 3.1·10-3 2.2·10-3 2.2·10-3 6.9·10-3 bPT (g ng-1) 0.036 0.11 0.24 0.20 0.20 0.30 sbPT (g ng-1) 1.1·10-3 6.4·10-3 4.4·10-3 7.0·10-3 7.0·10-3 1.1·10-2 tstudent 13 1 2 1 1 13 % R2 99.9 99.3 99.9 99.8 99.8 99.6 LOD (ng g-1) 1.5 2 0.5 0.03 0.03 0.03 LOQ (ng g-1) 4 6 2 0.08 0.08 0.08 LDR (ng g-1) 4.0-62.5 6.0-62.5 1.8-62.5 0.08-62.5 0.08-62.5 0.08-62.5 MP: Mobile phase; PT: Placental tissue; b: slope; sb: slope standard deviation; R2: determination coefficient; LOD: limit of detection; LOQ: limit of quantification; LDR: linear dynamic range Table Table 3 Recovery assay, precision and trueness of target compounds in placental tissue. Compound Spiked (ng g-1) Found (ng g-1) (SD) RSD (%) Recovery (%) PFBuA 6.25 6.02 (1) 14 96 12.5 12.8 (2) 12 103 25 28 (2) 6 113 PFPeA 6.25 6.61 (1) 9 106 12.5 12.3 (2) 10 98 25 24 (3) 9 94 PFHxA 6.25 6.44 (1) 10 103 12.5 12.4 (2) 12 99 25 23 (3) 12 94 PFHpA 1.25 1.32 (0.1) 1 105 6.25 6.24 (1) 4 100 12.5 11.8 (1) 12 95 PFOA 1.25 1.30 (0.1) 1 104 6.25 6.24 (1) 4 100 12.5 11.8 (1) 12 95 PFOS 1.25 1.30 (0.1) 1 104 6.25 6.11 (1) 13 98 12.5 12.6 (2) 14 101 SD: Standard deviation; RSD: Relative standard deviation Table Table 4 Concentrations of PFCs determined in the placenta samples. Sample *Found amount (ng g-1) (SD) PFBuA PFPeA PFHxA PFHpA PFOA PFOS Mother 01 D ND D ND D D Mother 02 ND ND ND ND ND ND Mother 03 ND ND ND ND ND ND Mother 04 ND ND D ND ND 1.1 (0.01) Mother 05 ND ND ND ND ND D Mother 06 ND ND ND ND ND D Mother 07 ND ND ND ND ND 1.0 (0.01) Mother 08 30 (2) ND 4.9 (0.5) ND D ND Mother 09 ND ND ND ND ND 1.2 (0.01) Mother 10 D ND D ND ND ND Mother 11 ND ND ND ND ND ND Mother 12 ND ND ND ND ND ND Mother 13 ND ND ND ND ND D Mother 14 ND ND D D D D Mother 15 28 (1.5) ND 5.1 (0.4) ND ND D Mother 16 ND ND D ND ND D Mother 17 ND ND ND ND ND ND Mother 18 ND ND D ND D 1.1 (0.01) Mother 19 ND ND ND ND ND ND Mother 20 D ND D ND ND D Mother 21 D ND ND D 0.37 (0.03) 1.2 (0.01) Mother 22 ND ND ND D ND ND Mother 23 ND ND ND ND ND ND Mother 24 ND ND ND D 0.37 (0.02) 0.21 (0.01) Mother 25 ND ND ND ND ND ND ND: below limit of detection; D: below the limit of quantification but above the limit of detection * Mean of six determinations Table 0 20 40 60 80 100 120 140 PFBuA PFPeA PFHxA PFHpA PFOA PFOS Recovery (%) Acetone Acetonitrile Ethyl acetate Figure Click here to download high resolution image Figure Click here to download high resolution image