TY - JOUR
AU - Waidyanatha, Suramya
AB - Abstract 2-Hydroxy-4-methoxybenzophenone (HMB) is a common ingredient in sunscreens and other personal care products and thus significant potential exists for human exposure. HMB was nominated to the National Toxicology Program (NTP) for testing due to its high exposure through consumer products and inadequate toxicological data at the time, which also included increasing concern for the potential effects of HMB on reproduction and development. HMB is metabolized to numerous metabolites in vivo and in vitro including 2,4-dihydroxybenzophenone (DHB), 2,3,4-trihydroxybenzophenone (THB) and 2,5-dihydroxy-4-methoxybenzophenone (2,5-DHMB) as well as their corresponding glucuronide and/or sulfate conjugates. In this study, we have developed and validated a liquid chromatography–tandem mass spectrometry method to quantitate free (unconjugated) HMB and DHB, and total (combined conjugated and unconjugated) HMB, DHB, THB and 2,5-DHMB. The method was successfully applied to quantitate these analytes in plasma from postnatal day 28 and 56 male and female Harlan Sprague Dawley rat pups following perinatal dietary exposure to 0 (control), 3,000, 10,000 and 30,000 ppm HMB beginning on gestational Day 6. All determined analyte concentrations increased with increasing dose and were significantly higher than the controls at both timepoints. All the total analytes were quantified in all plasma samples and total concentrations were considerably higher than free, suggesting extensive conjugation. Mean concentrations of total HMB and DHB were higher (~100–300-fold) than the free HMB and DHB concentrations, and total concentrations in plasma were approximately HMB≈DHB > 2,5-DHMB»THB. Free and total analyte plasma concentrations were not sex-dependent and in general, both free and total analytes were detected in the control samples. Comparison of our rat data, using the internal dose, with human data available in the literature suggests that the rat doses used in our studies were within 4-fold of the human dose. Introduction Personal care products containing ultraviolet (UV)-stabilizers, such as benzophenones, have been in use for several decades. 2-Hydroxy-4-methoxybenzophenone (HMB) is a benzophenone derivative and the most common component of sunscreen and other personal care products in the USA used to prevent UV light from passing through the skin. It is found in over 1,500 products at concentrations up to 0.15% (1, 2). Exposure to HMB occurs mainly via dermal and oral routes resulting from application of consumer products such as sunscreens and lip balms (3, 4). There are increasing concerns about the safety and toxicity of HMB following occupational exposure and via use of consumer product due to its incomplete toxicological profile. Recent studies also report concerns due to the transformation products of oxybenzones by chlorination during water treatment process (5–7) and kinetics (8, 9), stability and toxicity (10) of the transformation products. Potential adverse health effects associated with HMB exposure in in vivo and in vitro studies include reproductive and developmental toxicity which is hypothesized to occur via endocrine disruption (11–14). HMB was reported to exhibit estrogenic activity in in vitro (15, 16) and in vivo studies (13, 17). HMB is rapidly absorbed from the intact skin (3, 4, 18) and from gastrointestinal tract (19) and demethylated and excreted in the urine in humans (19, 20). HMB was detected in the urine of 97% general US population and the concentrations differed by socio-demographic factors (21, 22). Effects on birth weight and head circumference in humans were associated with high levels of HMB in the urine of mothers (23, 24). HMB and the metabolite 2,4-dihydroxybenzophenone (DHB) have shown some estrogenic and weakly anti-androgenic activity (25) and have been detected in human breast milk (20, 26, 27). In rats, higher plasma and urinary levels of HMB was detected in males compared to females have been reported and the difference was contributed to the possible gender differences in disposition and metabolism of HMB (22). HMB has been reported to metabolize DHB, 2,2′-dihydroxy-4-methoxybenzophenone (2,2′-DHMB) and 2,3,4-trihydroxybenzophenone (THB) in vivo (28–32) and in vitro (33). Additionally, 2,5-dihydroxy-4-methoxybenzophenone (2,5-DHMB) has been identified as a major metabolite formed by rat liver microsomes (34). 2,5-DHMB was also identified in rodents following oral exposure to HMB (Figure 1), however, contradictory to previously published reports, no 2,2′-DHMB was detected in any of the samples (NTP unpublished data). Figure 1. Open in new tabDownload slide 2-Hydroxy-4-methoxybenzophenone (HMB) and its free (DHB) and total metabolites (HMB, DHB, 2,3,4-trihydroxybenzophenone (THB) and 2,5-dihydroxy-4-methoxybenzophenone (2,5-DHMB)). Figure 1. Open in new tabDownload slide 2-Hydroxy-4-methoxybenzophenone (HMB) and its free (DHB) and total metabolites (HMB, DHB, 2,3,4-trihydroxybenzophenone (THB) and 2,5-dihydroxy-4-methoxybenzophenone (2,5-DHMB)). HMB was nominated to the National Toxicology Program (NTP) for toxicological characterization due to significant human exposure and lack of adequate data. Assessing systemic exposure to HMB and its metabolites following exposure is essential in explaining the toxicological outcome. There are several published methods to quantitate conjugated and unconjugated HMB and DHB in urine and plasma (26, 31, 35, 36). However, to the best of our knowledge, there are no methods to simultaneously quantitate free (unconjugated) and total (conjugated and unconjugated) HMB and its metabolites DHB, THB and 2,5-DHMB in rat plasma by liquid chromatography–tandem mass spectrometry (LC–MS-MS). The study reported here is a part of a larger study investigating the reproductive and developmental toxicity of HMB in HSD rats when administered via feed. The objective of this study is to develop and validate a LC–MS-MS method to simultaneously quantitate HMB and its metabolites DHB, THB and 2,5-DHMB in HSD rat plasma following perinatal dietary exposure and evaluate the metabolite formation differences with gender and age (juvenile, PND28 vs. adolescent, PND56) changes in support of NTP toxicology studies. Materials and Methods Chemicals and reagents HMB (CASRN 131-57-7) bulk was purchased from Ivy Fine Chemicals (Cherry Hill, NJ). Identity of the bulk chemical as HMB was confirmed by infrared spectrum and proton and carbon-13 nuclear magnetic resonance (NMR) spectroscopy. The purity was determined to be 100% by high differential scanning calorimetry. No reportable impurities were identified by high performance liquid chromatography (HPLC) with UV detection and by gas chromatography with flame ionization detection. HMB was confirmed to be stable during the conduct of the study. HMB, DHB (CASRN 131-56-6) and THB (CASRN 1143-72-2) standards were purchased from Sigma Aldrich (St. Louis, MO). 2,5-DHMB and 2,5-DHMB-d3 were purchased from Richman Chemicals (Loer Gwynedd, PA). HMB-d5, DHB-d5 and THB-d5 were purchased from C/D/N Isotopes, Inc. (Pointe-Claire, Quebec, Canada). Sprague Dawley rat plasma and HSD rat plasma for method validation were purchased from Bioreclamation IVT (New York, USA). Solid phase extraction (SPE) 96-well plates (Strata-X 33 um 60 mg polymeric reversed phase) were purchased from Phenomenex (Torrance, CA). Purified water (ASTM Type 1) was produced by an Aqua Solutions model 2121BL (Jaspers, Georgia) purification system. Methanol (HPLC grade), 1 mM ammonium acetate, Acetonitrile (HPLC Grade), formic acid (>98%), concentrated hydrochloric acid (HCl) and ethyl acetate (HPLC grade) were purchased from Sigma Aldrich (St. Louis, MO). KOH (ACS grade) was purchased from Fisher Scientific (Pittsburgh, PA). Preparation of solvent calibration standards, plasma calibration standards, and quality control samples Isotopically labeled HMB (HMB-d5), DHB (DHB-d5), THB (THB-d5) and 2,5-DHMB (2,5-DHMB-d3) were used as the internal standards (IS) for free and total analysis. Working IS solutions of HMB-d5 at 500 ng/mL for free and at 1,000 ng/mL for total, THB-d5 at 1,000 ng/mL, and DHB-d5 and 2,5-DHMB-d3 at 500 ng/mL were prepared in water. Stock solutions of HMB, DHB, THB and 2,5-DHMB were prepared in methanol at 500 and 1,000 μg/mL concentrations. Plasma calibration standards were prepared by spiking 950 μL blank pooled (male/female) Sprague Dawley rat plasma with 50 μL of the appropriate working standards prepared from two alternate stock solutions to obtain final plasma concentrations ranging from 15 to 150 ng/mL (free HMB and DHB), 200 to 2,000 ng/mL (total HMB and THB) or 40 to 400 ng/mL (total DHB and 2,5-DHMB). Solvent calibration standards were prepared similar to plasma calibration standards except no plasma was used. Plasma QC samples were prepared at three concentrations (low QC, mid QC and high QC) using an independent stock solution at concentrations; 20, 45, 110 ng/mL for free HMB and DHB, 250, 600, 1,500 ng/mL for total HMB and THB, and 50, 120 and 300 ng/mL for total DHB and 2,5-DHMB. Six replicates of each QC concentration were processed with each validation run. Preparation of plasma samples for analysis of free and total analytes For quantitation of free analytes, each well of a polymeric reversed phase SPE 96-well plate was conditioned with 50 μL of methanol followed by 500 μL of purified water. In total, 100 μL of each calibration standard, QC sample, plasma blank or study plasma samples were added to individual wells. Each well was spiked with 50 μL of working IS with the exception of the samples without IS where 50 μL of methanol/water (10:90 v/v) were added. For blanks without plasma, 50 μL of water was used. Each well was washed with 500 μL methanol/water (10:90 v/v). Samples were eluted with 2 × 500 μL2 of 0.1% formic acid/acetonitrile. The eluate was evaporated to dryness at 40°C under N2 and the residue was reconstituted in 100 μL of acetonitrile:1 mM aqueous ammonium acetate (20:80 v/v) for LC–MS-MS as given below. For quantitation of total analytes, 100 μL each calibration standard, QC sample, blank or study plasma samples were spiked with 50 μL of working IS, with the exception of the samples without IS. For blanks without plasma, 50 μL of water was used. Samples then were hydrolyzed with 200 μL of 6 N HCl at ∼ 100°C for ∼30 min. After bringing the samples to ambient temperature 150 μL of 6 N KOH was added, and samples were extracted with 600 μL of ethyl acetate. The supernatants were evaporated to dryness at ∼40°C under N2 and reconstituted in 150 μL methanol for LC–MS-MS analysis as given below. LC–MS-MS analysis Samples were analyzed using either Agilent 1,200 High Performance Liquid Chromatography (HPLC) (Santa Clara, CA) coupled to a Sciex API 4000Q (Toronto, Ontario Canada) mass spectrometer or Shimadzu Prominence HPLC (Kyoto, Japan) coupled to a Sciex Triple Quad 5,500 (Toronto, Ontario Canada) mass spectrometer. Transitions monitored were the m/z 227→211, 213→135, 229→151, 243→94 for HMB, DHB, THB and 2,5-DHMB, and m/z 232→215, 218→135, 234→151, 246→94 for internal standards HMB-d5, DHB-d5, THB-d5 and 2,5-DHMB-d3, respectively. Phenomenex Gemini-NX C18 column (50 × 2 mm2, 5 μ) (Torrance, CA) was used for analyte separation with a flow rate of 400 μL/min using the following gradient: (A) of 1 mM ammonium acetate/0.02% formic acid and (B) acetonitrile; 0–0.2 min, 20% B; 0.2–2.9 min 95% B, 2.9–4.0 min 95% B; 4.0–4.5 min 20% B; 4.5–9.5 min 20% B. The electrospray ionization interface was operated in negative ion mode. The mass spectrometer operating conditions were; ion spray voltage, 4,500 or 3,000 V; source temperature, 550 or 450°C; sheath and auxiliary gas pressures, 50 or 45 arbitrary units, collision energy, 30 eV. Analytical method validation Linearity, intra- and inter-day precision and accuracy of the method were evaluated by analyzing plasma calibration standards over the concentration range of 15–150 ng/mL for free HMB and DHB, 200–2,000 ng/mL for total HMB and THB, and 40–400 ng/mL for total 2,5-DHMB and DHB. Low, mid and high plasma quality control (QC) samples were also evaluated for intra- and inter-day precision and accuracy of the method. Dilution verification was conducted to evaluate whether the concentrations outside the validated range could be accurately quantitated after diluting into the range. Sensitivity of the methods was estimated by analyzing a set of six rat plasma QC samples prepared at the lower limit of quantitation (LLOQ) of 15 ng/mL for free HMB and THB, 40 ng/mL for total DHB and 2,5-DHMB, and 200 ng/mL for total HMB and THB. The limit of detection (LOD) was calculated as three times standard deviation of the LLOQ QC samples. Absolute recovery was calculated by comparing the ratio of spiked matrix standards to the ratio of the solvent standard. Relative recovery was calculated by comparing the response of spiked matrix standards to the response of the extracted plasma spiked with same amount of standards. Ruggedness was tested on two columns with different serial numbers and using two analysts over the course of the validation. Selectivity was checked by analyzing blank rat plasma samples without IS from six different sources. For carryover at least one blank rat plasma sample was analyzed immediately following a high standard. Long-term analyte and IS solvent storage stability was determined by analyzing four aliquots of solvent and matrix standards prepared at the LLOQ and stored for minimum of 60 days at ∼2–8°C by LC–MS-MS along with freshly prepared and processed solvent calibration standards and QC samples. Freeze/thaw-, short-term-, long-term-, and extended matrix stability of rat plasma QC samples was evaluated as follows. Freeze/thaw matrix stability was determined by analyzing four aliquots each of low, mid, and high rat plasma QC samples for free analytes, and of low, and high rat plasma QC samples for total analytes which had been stored at ~−70°C for at least 24 h, and undergone a minimum of three freeze/thaw cycles. Short-term matrix stability was determined by analyzing four aliquots each of low and high rat plasma QC samples which had been stored at ~−70°C for at least 24 h then stored at room temperature (RT) or refrigerated (2–8°C) for a minimum of 19 h. Long-term matrix stability was determined by analyzing four aliquots each of low and high QC samples which had been stored at ~−70°C for minimum of 60 days. Extended matrix stability was determined by analyzing four aliquots each of low and high QC samples which had been stored at ~−70°C for minimum of 980 days which covered the study sample storage period and conditions. Stability of analytes in extracted samples was evaluated up to 7 days at 2–8°C and reanalyzed for reinjection reproducibility by injecting the samples onto the LC–MS-MS system along with freshly prepared calibration standards. Animals, exposure and plasma collection Exposures to animals were conducted at RTI International (RTP, NC). All procedures that involved the use of animals were approved by RTI's Institutional Animal Care and Use Committee (IACUC). Time-mated, ∼gestation Day (GD) 2, female HSD rats (13–14 weeks; ~200–225 g) were purchased from Harlan Laboratories (Dublin, VA) and housed individually. They were provided tap water (city of Durham, NC) and milled feed ad libitum (irradiated and certified low phyto-estrogen 5K96 diet, PMI Nutrition International, St. Louis, MO). Prior to administration of HMB dosed feed, female rats were randomized into treatment groups and uniquely identified by tail tattoo. Pups were identified by paw tattoo according to number within litter at postnatal Day (PND) 1 and uniquely identified at weaning by tail tattoo. Dams were housed one per cage and pups remained with their respective dam until weaning on PND28. Twenty five time-mated female rats/group were exposed to HMB in 5K96 feed at 0 (control feed), 3,000, 10,000 and 30,000 ppm on GD6; exposure continued throughout gestation and lactation and until PND 58. Blood was collected from randomly selected animals (1/sex/litter) that had been exposed to HMB via 5K96 feed at the same dose levels as their respective dam until scheduled termination at PND28 or PND56. Prior to blood collection, the animals were euthanized by CO2 asphyxiation and blood was collected by cardiac puncture. Blood was centrifuged for 10 min at 270–360 g at 4°C within 1 h of collection and plasma was isolated and was stored at −80°C until analysis. Statistical analysis Values below the LOD were substituted with ½ the LOD for that analyte. Analyte levels were compared between dosed and control groups using the nonparametric multiple comparison methods of Shirley (37) and Dunn (38). Jonckheere's test (39) was used to assess the significance of dose-response trends and to determine whether a trend-sensitive test (Shirley's test) was more appropriate for pairwise comparisons than a test that does not assume a monotonic dose-response (Dunn's test). Trend-sensitive tests were used when Jonckheere's test was significant at P < 0.01. To take potential litter effects into account, the Wilcoxon Signed Rank Test was used to compare analyte levels between males and females. Only data with both male and female pups from the same dam were included in this analysis. The exact Wilcoxon Two-Sample Rank Sum Test was used to compare analyte levels at PND28 with PND56. These analyses were conducted separately for each dose group and Bonferroni corrections were used to adjust for multiple comparisons. Comparisons of Free DHB with Free HMB, Free DHB with Total DHB and Free HMB with Total HMB were done on the ranked data using multivariate analysis of variance, with within-subjects contrasts comparing the two metabolites within each dose group (40). A Bonferroni correction was used to adjust for multiple comparisons. Values that fell below LOD of either endpoint were substituted with ½ the larger LOD. Only animals having measurements on both endpoints were included in this analysis. Results Analytical method validation The spiked samples calibration regression model for free HMB and DHB was quadratic and for total HMB, DHB, THB and 2,5-DHMB was linear with a weight factor of 1/x for free and total analytes. The plasma standard curves had coefficient of determination (r2) values ≥0.98 for free and total analytes in all accepted runs. The intra- and inter-day accuracy and precision of plasma calibration standards and quality control (QC) samples were determined as relative error (RE%) percent and relative standard derivation (RSD). The intra- and inter-day precision met the acceptance criteria with average RE% within 15% of nominal concentration (≤ ± 10.1%) and RSD values ≤15% for both free and total analytes except at the LLOQ for free DHB and total HMB, which RSDs were 27.2 and 29.6%, respectively (Table I). The QC samples also met accuracy and precision acceptance criteria in all runs for both free and total analytes with the RE% ≤ ±15% of nominal concentration (≤ ± 13.8%) except at low QC for total THB (≤ ± 17.5%) and RSD values were within ≤15% (≤ ± 11.6%) except at high QC for total DHB, which was 28.5% for intra-day and 16.5% for inter-day (Table I). Table I. Method validation and stability data for free and total analyses . Free . Total . HMB . DHB . HMB . DHB . THB . 2,5-DHMB . Matrix concentration range (ng/mL) 15–150 15–150 200–2,000 40–400 200–2,000 40–400 LOD (ng/mL) 0.648 6.22 68.7 1.62 81.9 11.0 LLOQa (ng/mL) 15 15 200 40 200 40 Accuracy (RE %)  Intra-dayb ≤ ± 5.4 ≤ ± 6.9 ≤ ± 10.1 ≤ ± 5.6 ≤ ± 5.0 ≤ ± 8.1  Inter-dayb ≤ ± 3.4 ≤ ± 3.2 ≤ ± 7.0 ≤ ± 3.4 ≤ ± 2.4 ≤ ± 4.2  Intra-dayc ≤ ± 8.1 ≤ ± 13.8 ≤ ± 4.9 ≤ ± 12.8 ≤ ± 17.5 ≤ ± 8.2  Inter-dayc ≤ ± 6.3 ≤ ± 11.0 ≤ ± −3.1 ≤ ± 3.9 ≤ ± 11.1 ≤ ± 2.5 Precision RSD (%)  Intra-dayb ≤10.8 ≤27.2 ≤29.6 ≤2.7 ≤9.0 ≤12.5  Inter-dayb ≤5.0 ≤14.4 ≤14.4 ≤3.0 ≤4.9 ≤4.2  Intra-dayc ≤3.9 ≤10.8 ≤11.6 ≤28.5 ≤4.5 ≤8.6  Inter-dayc ≤3.9 ≤8.1 ≤10.4 ≤16.3 ≤10.2 ≤7.4 Stability (RE %)  Freeze–thaw matrix −3.0 to 1.7 −8.2 to 5.0 −1.1 to 0.0 0.3 to 3.5 NS NS  Short-term matrix (RT) −3.5 to 1.7 −4.8 to 7.5 1.3 to 1.9 −2.4 to −0.2 NS NS  Long-term matrix (−70°C) −6.0 to 5.9 −1.3 to 13.6 2.3 to 2.5 3.5 to 6.4 −9.8 to −8.5 11.0 to 18.8  Extended matrix (−70°C) ND ND −5.7 to 18.3 −4.5 to 2.7 −36.5 to −22.2 −20.5 to −79.0  Extract 2–8 s°C ND ND ≤123.3 ≤52.3 ≤63.6 ≤60.5 Recovery %  Absolute 213 117 80c 71.1c 62.9c 65.1c  Relative 95.6 88.4 92.3 91.6 93.2 91.0  Reinjection reproducibility (RE%) ≤4.1 ≤4.1 ≤13 ≤6.3 ≤2.4 ≤5.7 . Free . Total . HMB . DHB . HMB . DHB . THB . 2,5-DHMB . Matrix concentration range (ng/mL) 15–150 15–150 200–2,000 40–400 200–2,000 40–400 LOD (ng/mL) 0.648 6.22 68.7 1.62 81.9 11.0 LLOQa (ng/mL) 15 15 200 40 200 40 Accuracy (RE %)  Intra-dayb ≤ ± 5.4 ≤ ± 6.9 ≤ ± 10.1 ≤ ± 5.6 ≤ ± 5.0 ≤ ± 8.1  Inter-dayb ≤ ± 3.4 ≤ ± 3.2 ≤ ± 7.0 ≤ ± 3.4 ≤ ± 2.4 ≤ ± 4.2  Intra-dayc ≤ ± 8.1 ≤ ± 13.8 ≤ ± 4.9 ≤ ± 12.8 ≤ ± 17.5 ≤ ± 8.2  Inter-dayc ≤ ± 6.3 ≤ ± 11.0 ≤ ± −3.1 ≤ ± 3.9 ≤ ± 11.1 ≤ ± 2.5 Precision RSD (%)  Intra-dayb ≤10.8 ≤27.2 ≤29.6 ≤2.7 ≤9.0 ≤12.5  Inter-dayb ≤5.0 ≤14.4 ≤14.4 ≤3.0 ≤4.9 ≤4.2  Intra-dayc ≤3.9 ≤10.8 ≤11.6 ≤28.5 ≤4.5 ≤8.6  Inter-dayc ≤3.9 ≤8.1 ≤10.4 ≤16.3 ≤10.2 ≤7.4 Stability (RE %)  Freeze–thaw matrix −3.0 to 1.7 −8.2 to 5.0 −1.1 to 0.0 0.3 to 3.5 NS NS  Short-term matrix (RT) −3.5 to 1.7 −4.8 to 7.5 1.3 to 1.9 −2.4 to −0.2 NS NS  Long-term matrix (−70°C) −6.0 to 5.9 −1.3 to 13.6 2.3 to 2.5 3.5 to 6.4 −9.8 to −8.5 11.0 to 18.8  Extended matrix (−70°C) ND ND −5.7 to 18.3 −4.5 to 2.7 −36.5 to −22.2 −20.5 to −79.0  Extract 2–8 s°C ND ND ≤123.3 ≤52.3 ≤63.6 ≤60.5 Recovery %  Absolute 213 117 80c 71.1c 62.9c 65.1c  Relative 95.6 88.4 92.3 91.6 93.2 91.0  Reinjection reproducibility (RE%) ≤4.1 ≤4.1 ≤13 ≤6.3 ≤2.4 ≤5.7 aLower limit of quantitation (LLOQ) is the lowest concentration of the calibration curve. ND, not determined; NS, not stable. bEstimated based on plasma calibration standards. cEstimated based on low, mid and high QC samples. Open in new tab Table I. Method validation and stability data for free and total analyses . Free . Total . HMB . DHB . HMB . DHB . THB . 2,5-DHMB . Matrix concentration range (ng/mL) 15–150 15–150 200–2,000 40–400 200–2,000 40–400 LOD (ng/mL) 0.648 6.22 68.7 1.62 81.9 11.0 LLOQa (ng/mL) 15 15 200 40 200 40 Accuracy (RE %)  Intra-dayb ≤ ± 5.4 ≤ ± 6.9 ≤ ± 10.1 ≤ ± 5.6 ≤ ± 5.0 ≤ ± 8.1  Inter-dayb ≤ ± 3.4 ≤ ± 3.2 ≤ ± 7.0 ≤ ± 3.4 ≤ ± 2.4 ≤ ± 4.2  Intra-dayc ≤ ± 8.1 ≤ ± 13.8 ≤ ± 4.9 ≤ ± 12.8 ≤ ± 17.5 ≤ ± 8.2  Inter-dayc ≤ ± 6.3 ≤ ± 11.0 ≤ ± −3.1 ≤ ± 3.9 ≤ ± 11.1 ≤ ± 2.5 Precision RSD (%)  Intra-dayb ≤10.8 ≤27.2 ≤29.6 ≤2.7 ≤9.0 ≤12.5  Inter-dayb ≤5.0 ≤14.4 ≤14.4 ≤3.0 ≤4.9 ≤4.2  Intra-dayc ≤3.9 ≤10.8 ≤11.6 ≤28.5 ≤4.5 ≤8.6  Inter-dayc ≤3.9 ≤8.1 ≤10.4 ≤16.3 ≤10.2 ≤7.4 Stability (RE %)  Freeze–thaw matrix −3.0 to 1.7 −8.2 to 5.0 −1.1 to 0.0 0.3 to 3.5 NS NS  Short-term matrix (RT) −3.5 to 1.7 −4.8 to 7.5 1.3 to 1.9 −2.4 to −0.2 NS NS  Long-term matrix (−70°C) −6.0 to 5.9 −1.3 to 13.6 2.3 to 2.5 3.5 to 6.4 −9.8 to −8.5 11.0 to 18.8  Extended matrix (−70°C) ND ND −5.7 to 18.3 −4.5 to 2.7 −36.5 to −22.2 −20.5 to −79.0  Extract 2–8 s°C ND ND ≤123.3 ≤52.3 ≤63.6 ≤60.5 Recovery %  Absolute 213 117 80c 71.1c 62.9c 65.1c  Relative 95.6 88.4 92.3 91.6 93.2 91.0  Reinjection reproducibility (RE%) ≤4.1 ≤4.1 ≤13 ≤6.3 ≤2.4 ≤5.7 . Free . Total . HMB . DHB . HMB . DHB . THB . 2,5-DHMB . Matrix concentration range (ng/mL) 15–150 15–150 200–2,000 40–400 200–2,000 40–400 LOD (ng/mL) 0.648 6.22 68.7 1.62 81.9 11.0 LLOQa (ng/mL) 15 15 200 40 200 40 Accuracy (RE %)  Intra-dayb ≤ ± 5.4 ≤ ± 6.9 ≤ ± 10.1 ≤ ± 5.6 ≤ ± 5.0 ≤ ± 8.1  Inter-dayb ≤ ± 3.4 ≤ ± 3.2 ≤ ± 7.0 ≤ ± 3.4 ≤ ± 2.4 ≤ ± 4.2  Intra-dayc ≤ ± 8.1 ≤ ± 13.8 ≤ ± 4.9 ≤ ± 12.8 ≤ ± 17.5 ≤ ± 8.2  Inter-dayc ≤ ± 6.3 ≤ ± 11.0 ≤ ± −3.1 ≤ ± 3.9 ≤ ± 11.1 ≤ ± 2.5 Precision RSD (%)  Intra-dayb ≤10.8 ≤27.2 ≤29.6 ≤2.7 ≤9.0 ≤12.5  Inter-dayb ≤5.0 ≤14.4 ≤14.4 ≤3.0 ≤4.9 ≤4.2  Intra-dayc ≤3.9 ≤10.8 ≤11.6 ≤28.5 ≤4.5 ≤8.6  Inter-dayc ≤3.9 ≤8.1 ≤10.4 ≤16.3 ≤10.2 ≤7.4 Stability (RE %)  Freeze–thaw matrix −3.0 to 1.7 −8.2 to 5.0 −1.1 to 0.0 0.3 to 3.5 NS NS  Short-term matrix (RT) −3.5 to 1.7 −4.8 to 7.5 1.3 to 1.9 −2.4 to −0.2 NS NS  Long-term matrix (−70°C) −6.0 to 5.9 −1.3 to 13.6 2.3 to 2.5 3.5 to 6.4 −9.8 to −8.5 11.0 to 18.8  Extended matrix (−70°C) ND ND −5.7 to 18.3 −4.5 to 2.7 −36.5 to −22.2 −20.5 to −79.0  Extract 2–8 s°C ND ND ≤123.3 ≤52.3 ≤63.6 ≤60.5 Recovery %  Absolute 213 117 80c 71.1c 62.9c 65.1c  Relative 95.6 88.4 92.3 91.6 93.2 91.0  Reinjection reproducibility (RE%) ≤4.1 ≤4.1 ≤13 ≤6.3 ≤2.4 ≤5.7 aLower limit of quantitation (LLOQ) is the lowest concentration of the calibration curve. ND, not determined; NS, not stable. bEstimated based on plasma calibration standards. cEstimated based on low, mid and high QC samples. Open in new tab Sensitivity of the method was determined by analyzing the plasma LLOQ QC samples prepared from six different rat plasma lots which met accuracy and precision acceptance criteria with average determined concentrations being ≤ ± 20% of nominal and RSD ≤20% for both free and total methods (Table I). The estimated LOD for free and total analytes are represented in Table I. The rat plasma dilution QC samples met accuracy and precision acceptance criteria (average determined concentrations ≤ ± 15% of nominal and RSD values ≤15%, Table I). Standards, as high as 60 μg/mL for free HMB and DHB and 175 μg/mL for total HMB, DHB, THB and 2,5-DHMB, could successfully be diluted with plasma into the validated concentration range. The absolute recovery of the total analytes ranged from 62.9 to 80.0% while relative recoveries for free and total analytes ranged from 88.4 to 95.6% (Table I). Selectivity samples from six different sources were processed without IS and evaluated for the response of any peak with retention time of the analytes or IS with >30% of the average response of the LLOQ standards. There were no significant interfering peaks (>30% of LLOQ) at the approximate retention time of the free and total analytes or internal standards (Figure 2). No peaks were found at the quantitative window of the analyte or IS that would cause interference. Figure 2. Open in new tabDownload slide Selected ion chromatograms for HMB, DHB, THB, 2,5-DHMB from a male PND56 plasma following exposure to 0 (control), 3,000, 30,000 ppm HMB via feed. Figure 2. Open in new tabDownload slide Selected ion chromatograms for HMB, DHB, THB, 2,5-DHMB from a male PND56 plasma following exposure to 0 (control), 3,000, 30,000 ppm HMB via feed. Carryover was evaluated by injecting a rat plasma blank immediately following an extracted high standard injection and there was no evidence of carryover for the free and total analytes or IS in the validation runs. The area response in the carryover blank sample compared to that of the nearest low standard met the acceptance criteria of ≤30%. Reinjection reproducibility met acceptance criteria with the average determined concentration of the QC samples ≤ ± 15% of nominal and RSD values ≤15% at each concentration level upon reinjection (Table I). The results indicate that an entire set of rat plasma extracts, including standards and QCs extracted on the same day as the samples, can be reinjected when stored at the temperature of the autosampler (room temperature) for up to 7 days. A run set of ~100 rat validation sample injections conducted by two different analysts and using two different HPLC columns produced equivalent data that met acceptance criteria. Stability of the analytes and IS in the extracted samples were evaluated by storing four aliquots of plasma samples prepared at the LLOQ concentrations at 2–8°C protected from light for a minimum of 60 days and analyzing them at the end of the storage length. Analytes were found to be stable for 70 days for free method and 66 days for total method with concentrations ≤15% of the nominal concentrations (RSD ≤ 12.2%) (Table I). HMB-d5, DHB-d5, THB-d5 and 2,5-DHMB-d3 IS in extracts were ≤ ±15% of the nominal concentration and were stable up to 55 days when stored at 2–8°C protected from light. Freeze–thaw plasma (matrix) storage stability was performed through four freeze–thaw cycles (−70°C/RT) protected from light. Precision (RSD ≤ 15%) and accuracy (RE% ≤ ±15%) for free and total HMB and DHB met the acceptance criteria (RSD ≤ 7.5%, RE% ≤ ±8.2%), while total THB and 2,5-DHMB met only precision (RSD ≤4.7%) but not the accuracy (RE% ≤ ±83.4%) acceptance criteria. Short-term and long-term matrix storage stability of the analytes was evaluated at low and high QC concentrations. Short-term stability was performed after 24 h of storage at ~−70°C. The results met acceptance criteria (RE% ≤ ±15% and RSD ≤ 15%) for HMB (RE% ≤ ±4.4% and RSD ≤ 10.7%) and DHB (RE% ≤ ±7.5% and RSD ≤ 12.4%), while THB (RE% ≤ ±92.4% and RSD ≤ 5.1%) and 2,5-DHMB (RE% ≤ ±95.4% and RSD ≤40%) were found to be outside of the acceptance criteria under the same conditions (Table I). Long-term stability was performed after minimum of 60 days of storage at ~−70°C. The results met acceptance criteria (RE% ≤ ±15% and RSD ≤ 15%) for all analytes (RE% ≤ ±13.6% and RSD ≤ 10.5%) with the exception of 2,5-DHMB with slightly less accuracy (RE% ≤ ±18.8%). Extended plasma (matrix) storage stability was evaluated by analyzing low and high plasma QC samples of the analytes in rat plasma after storage at −70°C for up to 994 days to cover the study sample storage duration. HMB and DHB were stable for up to 994 days as demonstrated by a RE% ≤ ±15.0%. However, RE% for THB were −36.5 to −22.2 and for 2,5-DHMB were −20.5 to −79.0%, for low and high QC samples. For 2,5-DHMB, the high RE% observed for high QC was unexpected and likely due to multiple factors including inherent instability due to presence of multiple hydroxyl groups and/or analytical method variability at the time of analysis. Extract stability was evaluated and it was found that the rat plasma extract samples could not be reinjected and analyzed with freshly extracted calibration standard when stored at the temperature of the autosampler (2–8°C) for up to 7 days. The extract stability results for total analytes had >15% of the nominal concentrations and RSD ≤ 15% and did not meet the acceptance criteria (Table I). Analysis of plasma from PND28 and 56 male and female rats following perinatal exposure to HMB Validated methods for the analysis of free and total HMB were applied to study plasma samples (n = 4–5 per dose group and sex) of male and female rats following exposure to 0, 3,000, 10,000, and 30,000 ppm HMB via feed. Seven-point matrix calibration curves, from 15 to 150 ng/mL for free HMB and DHB, from 200 to 2,000 ng/mL for total HMB and THB and from 40 to 400 ng/mL for total DHB and 2,5-DHMB, were run at the beginning and end of each sample run. The performance of the calibration curve was evaluated prior to the analysis of each sample set. A successful calibration was indicated by the following parameters: coefficient of determination r2 ≥ 0.98 and RE% ≤ ± 15% for the standards (except at LLOQ RE% ≤ ± 20%). The plasma standard curves for free and total analytes had coefficient of determination (r2) values ≥0.98, in all accepted runs. Data from study samples were considered valid if they were the interspersed QC set was acceptable. Each sample set, method blanks and controls contained a interspersed QC set, which consisted of two concentrations of calibration standards prepared at the low and high ends of the calibration curve, with the number of QC standards prepared at each concentration corresponding to ≥10% of total samples analyzed per analysis day. A QC set passed when the measured concentration for at least 67% of the QC standards overall and at least 50% of the QC standards at each concentration level were within 15% of the nominal values. If the QC standards failed, it was necessary to reanalyze the samples. To determine reproducibility of analysis, ≥10% of samples were repeated. The concentration of each analyte was calculated using response ratio of analyte to corresponding IS, the regression equation, sample volume and dilution when applicable. The concentration of analytes in plasma was expressed as ng/mL. Each method was selective for both free and total analytes and intra- and inter-day precision and accuracy for free and total analytes met the acceptance criteria (Table I). All samples whose concentrations were below the LOQ have estimated concentration values below the LLOQ. The BLOD was used for the samples with below LOD values. The feed consumption date for PND28 was available as combined dam and pup and hence pup PND28 plasma concentration data was not adjusted for feed consumption; adjustment could be only made to PND56 data. Free THB and DHMB was not detected during preliminary analysis of study samples and hence these analytes were not included during the main analysis. Both free HMB and DHB were detected in all exposed animals on PND28 and PND56. Free HMB and DHB concentrations increased with dose in both male and female PND28 and PND56 plasma samples and the concentrations of the two analytes were statistically similar (Table II and Figure 3). Free DHB concentrations were not significantly higher than free HMB concentrations (Table II). No significant sex differences were found between PND28 or PND56 for both analytes at all exposure concentration examined. Only at 10,000 ppm, both analytes were significantly higher in PND56 than PND28 in both sexes (P ≤ 0.05). Both analytes were detected in some of the control samples; however, the levels were significantly lower than the lowest exposed group (3,000 ppm) concentrations. Table II. Free HMB and DHB concentrations in PND28 and PND56 male and female rats (n = 4–5) following perinatal exposure to HMB via feed Analyte . Age . Sex . Dose (ppm) . 0 (ng/mL) Mean ± SE . 3,000 (ng/mL) Mean ± SE . 10,000 (ng/mL) Mean ± SE . 30,000 (ng/mL) Mean ± SE . HMB PND28 M 3.97 ± 2.06 14.07 ± 1.63 44.98 ± 15.55 94.10 ± 21.98 F 2.29 ± 0.79 18.50 ± 4.95 36.08 ± 4.87 171.88 ± 47.83 PND56 M 1.63 ± 0.18 55.10 ± 5.70 188.40 ± 19.51 a 335.60 ± 59.85 F 1.38 ± 0.18 20.80 ± 3.11 98.56 ± 12.35 a 185.00 ± 36.17 DHB PND28 M 6.84 ± 2.16 35.3 ± 3.35 61.68 ± 14.51 89.85 ± 18.86 F 6.09 ± 1.88 32.60 ± 2.43 64.10 ± 9.65 111.58 ± 14.72 PND56 M 3.11 ± 0.00 152.30 ± 52.50 209.00 ± 34.90 a 301.00 ± 64.88 F 3.11 ± 0.00 95.00 ± 18.93 172.20 ± 22.11 a 224.80 ± 26.27 Analyte . Age . Sex . Dose (ppm) . 0 (ng/mL) Mean ± SE . 3,000 (ng/mL) Mean ± SE . 10,000 (ng/mL) Mean ± SE . 30,000 (ng/mL) Mean ± SE . HMB PND28 M 3.97 ± 2.06 14.07 ± 1.63 44.98 ± 15.55 94.10 ± 21.98 F 2.29 ± 0.79 18.50 ± 4.95 36.08 ± 4.87 171.88 ± 47.83 PND56 M 1.63 ± 0.18 55.10 ± 5.70 188.40 ± 19.51 a 335.60 ± 59.85 F 1.38 ± 0.18 20.80 ± 3.11 98.56 ± 12.35 a 185.00 ± 36.17 DHB PND28 M 6.84 ± 2.16 35.3 ± 3.35 61.68 ± 14.51 89.85 ± 18.86 F 6.09 ± 1.88 32.60 ± 2.43 64.10 ± 9.65 111.58 ± 14.72 PND56 M 3.11 ± 0.00 152.30 ± 52.50 209.00 ± 34.90 a 301.00 ± 64.88 F 3.11 ± 0.00 95.00 ± 18.93 172.20 ± 22.11 a 224.80 ± 26.27 aSignificantly higher than PND28 (P < 0.05). Open in new tab Table II. Free HMB and DHB concentrations in PND28 and PND56 male and female rats (n = 4–5) following perinatal exposure to HMB via feed Analyte . Age . Sex . Dose (ppm) . 0 (ng/mL) Mean ± SE . 3,000 (ng/mL) Mean ± SE . 10,000 (ng/mL) Mean ± SE . 30,000 (ng/mL) Mean ± SE . HMB PND28 M 3.97 ± 2.06 14.07 ± 1.63 44.98 ± 15.55 94.10 ± 21.98 F 2.29 ± 0.79 18.50 ± 4.95 36.08 ± 4.87 171.88 ± 47.83 PND56 M 1.63 ± 0.18 55.10 ± 5.70 188.40 ± 19.51 a 335.60 ± 59.85 F 1.38 ± 0.18 20.80 ± 3.11 98.56 ± 12.35 a 185.00 ± 36.17 DHB PND28 M 6.84 ± 2.16 35.3 ± 3.35 61.68 ± 14.51 89.85 ± 18.86 F 6.09 ± 1.88 32.60 ± 2.43 64.10 ± 9.65 111.58 ± 14.72 PND56 M 3.11 ± 0.00 152.30 ± 52.50 209.00 ± 34.90 a 301.00 ± 64.88 F 3.11 ± 0.00 95.00 ± 18.93 172.20 ± 22.11 a 224.80 ± 26.27 Analyte . Age . Sex . Dose (ppm) . 0 (ng/mL) Mean ± SE . 3,000 (ng/mL) Mean ± SE . 10,000 (ng/mL) Mean ± SE . 30,000 (ng/mL) Mean ± SE . HMB PND28 M 3.97 ± 2.06 14.07 ± 1.63 44.98 ± 15.55 94.10 ± 21.98 F 2.29 ± 0.79 18.50 ± 4.95 36.08 ± 4.87 171.88 ± 47.83 PND56 M 1.63 ± 0.18 55.10 ± 5.70 188.40 ± 19.51 a 335.60 ± 59.85 F 1.38 ± 0.18 20.80 ± 3.11 98.56 ± 12.35 a 185.00 ± 36.17 DHB PND28 M 6.84 ± 2.16 35.3 ± 3.35 61.68 ± 14.51 89.85 ± 18.86 F 6.09 ± 1.88 32.60 ± 2.43 64.10 ± 9.65 111.58 ± 14.72 PND56 M 3.11 ± 0.00 152.30 ± 52.50 209.00 ± 34.90 a 301.00 ± 64.88 F 3.11 ± 0.00 95.00 ± 18.93 172.20 ± 22.11 a 224.80 ± 26.27 aSignificantly higher than PND28 (P < 0.05). Open in new tab Figure 3. Open in new tabDownload slide Free HMB (A) and DHB (B) concentrations in PND28 and PND56 male and female rat plasma following exposure to 0 (control), 3,000, 30,000 ppm HMB via feed. Figure 3. Open in new tabDownload slide Free HMB (A) and DHB (B) concentrations in PND28 and PND56 male and female rat plasma following exposure to 0 (control), 3,000, 30,000 ppm HMB via feed. All four of the total analytes were detected in all exposed animals on PND28 and PND56. In general, total concentrations in plasma were approximately HMB ≈ DHB > 2,5-DHMB » THB (Table III). For HMB and DHB, total concentrations were significantly higher than respective free HMB and DHB concentrations (Table III). Similar to the free analysis, concentrations of total analytes increased with increasing dose at PND28 and PND56 in both sexes (Figure 4) with no significant sex difference. At PND56, total DHB, THB, and 2,5-DHMB in 10,000 ppm males and in 3,000 and 10,000 ppm in females were significantly higher (P < 0.05) than corresponding PND28 groups, suggesting higher overall exposure at PND56 in those dose groups. Total HMB concentrations were significantly higher in male rats from the 10,000 ppm (P < 0.05). All total analytes were detected in all controls at PND28 and in some at PND56; however, the levels were significantly (P < 0.05 or P < 0.01) lower than the lowest exposed group (Table III and Figure 4). Table III. Total HMB and metabolite concentrations in PND28 and PND56 male and female rats (n = 4–5) following perinatal exposure to HMB via feed Analyte . Age . Sex . Dose (ppm) . 0 (ng/mL) Mean ± SE . 3,000 (ng/mL) Mean ± SE . 10,000 (ng/mL) Mean ± SE . 30,000 (ng/mL) Mean ± SE . HMB PND28 M 590.9 ± 321.9a 3,470.0 ± 1,051.9b 8,822.0 ± 2,781.5b 16,940.0 ± 5498.9b F 352.0 ± 211.0c 2,080.0 ± 156.9b 7,694.0 ± 2501.4b 16,703.3 ± 6908.5c PND56 M 81.7 ± 2.7b 10,600.0 ± 1495.6b 40,100.0 ± 1499.0b,d 5,7900.0 ± 8494.8a F 79.82 ± 2.42b 2,572.0 ± 369.7b 12,204.0 ± 1584.9b 43,350.0 ± 7468.2b DHB PND28 M 1,147.8 ± 723.9 7,941.0 ± 1868.1a 13,182.0 ± 2498.9b 25,260.0 ± 5044.8b F 776.5 ± 613.5c 5,850.0 ± 542.3b 13,758.0 ± 1823.6b 20,636.7 ± 7998.9c PND56 M 0.8 ± 0.0 21,526.0 ± 4205.2b 40,840.0 ± 6861.7b,d 56,250.0 ± 6121.8b F 20.1 ± 0.0 14,006.0 ± 2701.3b,d 25,460.0 ± 3176.1b,d 48,875.0 ± 6316.8b THB PND28 M 183.0 ± 61.0 1,463.3 ± 141.9 2,614.0 ± 380.7 7,908.0 ± 1394.0 F 166.0 ± 68.0 1,173.3 ± 17.6 3,744.0 ± 292.3 5,740.0 ± 1924.3 PND56 M 40.9 ± 0.0 2,830.0 ± 247.9 8,146.0 ± 737.4d 14,037.5 ± 3006.1 F 40.9 ± 0.0 3,160.0 ± 392.2d 11,696.0 ± 800.4d 17,050.0 ± 2554.2 2,5-DHMB PND28 M 907.0 ± 530.3 5,496.7 ± 1102.9 11,860.0 ± 1546.5 20,300.0 ± 2805.9 F 528.0 ± 410.0 4,900.0 ± 680.1 11,580.0 ± 1339.2 19,660.0 ± 7940.6 PND56 M 20.0 ± 0.0 7,924.0 ± 959.3 25,740.0 ± 1202.7d 48,400.0 ± 9493.2 F 20.0 ± 0.0 9,484.0 ± 1325.2d 29,640.0 ± 2903.9d 36,725.0 ± 2101.7 Analyte . Age . Sex . Dose (ppm) . 0 (ng/mL) Mean ± SE . 3,000 (ng/mL) Mean ± SE . 10,000 (ng/mL) Mean ± SE . 30,000 (ng/mL) Mean ± SE . HMB PND28 M 590.9 ± 321.9a 3,470.0 ± 1,051.9b 8,822.0 ± 2,781.5b 16,940.0 ± 5498.9b F 352.0 ± 211.0c 2,080.0 ± 156.9b 7,694.0 ± 2501.4b 16,703.3 ± 6908.5c PND56 M 81.7 ± 2.7b 10,600.0 ± 1495.6b 40,100.0 ± 1499.0b,d 5,7900.0 ± 8494.8a F 79.82 ± 2.42b 2,572.0 ± 369.7b 12,204.0 ± 1584.9b 43,350.0 ± 7468.2b DHB PND28 M 1,147.8 ± 723.9 7,941.0 ± 1868.1a 13,182.0 ± 2498.9b 25,260.0 ± 5044.8b F 776.5 ± 613.5c 5,850.0 ± 542.3b 13,758.0 ± 1823.6b 20,636.7 ± 7998.9c PND56 M 0.8 ± 0.0 21,526.0 ± 4205.2b 40,840.0 ± 6861.7b,d 56,250.0 ± 6121.8b F 20.1 ± 0.0 14,006.0 ± 2701.3b,d 25,460.0 ± 3176.1b,d 48,875.0 ± 6316.8b THB PND28 M 183.0 ± 61.0 1,463.3 ± 141.9 2,614.0 ± 380.7 7,908.0 ± 1394.0 F 166.0 ± 68.0 1,173.3 ± 17.6 3,744.0 ± 292.3 5,740.0 ± 1924.3 PND56 M 40.9 ± 0.0 2,830.0 ± 247.9 8,146.0 ± 737.4d 14,037.5 ± 3006.1 F 40.9 ± 0.0 3,160.0 ± 392.2d 11,696.0 ± 800.4d 17,050.0 ± 2554.2 2,5-DHMB PND28 M 907.0 ± 530.3 5,496.7 ± 1102.9 11,860.0 ± 1546.5 20,300.0 ± 2805.9 F 528.0 ± 410.0 4,900.0 ± 680.1 11,580.0 ± 1339.2 19,660.0 ± 7940.6 PND56 M 20.0 ± 0.0 7,924.0 ± 959.3 25,740.0 ± 1202.7d 48,400.0 ± 9493.2 F 20.0 ± 0.0 9,484.0 ± 1325.2d 29,640.0 ± 2903.9d 36,725.0 ± 2101.7 aSignificantly higher than the free metabolite (P < 0.05). bSignificantly higher than the free metabolite (P < 0.01). cComparison was not available due to having only two non-missing pairs. dSignificantly higher than PND28 (P < 0.05). Open in new tab Table III. Total HMB and metabolite concentrations in PND28 and PND56 male and female rats (n = 4–5) following perinatal exposure to HMB via feed Analyte . Age . Sex . Dose (ppm) . 0 (ng/mL) Mean ± SE . 3,000 (ng/mL) Mean ± SE . 10,000 (ng/mL) Mean ± SE . 30,000 (ng/mL) Mean ± SE . HMB PND28 M 590.9 ± 321.9a 3,470.0 ± 1,051.9b 8,822.0 ± 2,781.5b 16,940.0 ± 5498.9b F 352.0 ± 211.0c 2,080.0 ± 156.9b 7,694.0 ± 2501.4b 16,703.3 ± 6908.5c PND56 M 81.7 ± 2.7b 10,600.0 ± 1495.6b 40,100.0 ± 1499.0b,d 5,7900.0 ± 8494.8a F 79.82 ± 2.42b 2,572.0 ± 369.7b 12,204.0 ± 1584.9b 43,350.0 ± 7468.2b DHB PND28 M 1,147.8 ± 723.9 7,941.0 ± 1868.1a 13,182.0 ± 2498.9b 25,260.0 ± 5044.8b F 776.5 ± 613.5c 5,850.0 ± 542.3b 13,758.0 ± 1823.6b 20,636.7 ± 7998.9c PND56 M 0.8 ± 0.0 21,526.0 ± 4205.2b 40,840.0 ± 6861.7b,d 56,250.0 ± 6121.8b F 20.1 ± 0.0 14,006.0 ± 2701.3b,d 25,460.0 ± 3176.1b,d 48,875.0 ± 6316.8b THB PND28 M 183.0 ± 61.0 1,463.3 ± 141.9 2,614.0 ± 380.7 7,908.0 ± 1394.0 F 166.0 ± 68.0 1,173.3 ± 17.6 3,744.0 ± 292.3 5,740.0 ± 1924.3 PND56 M 40.9 ± 0.0 2,830.0 ± 247.9 8,146.0 ± 737.4d 14,037.5 ± 3006.1 F 40.9 ± 0.0 3,160.0 ± 392.2d 11,696.0 ± 800.4d 17,050.0 ± 2554.2 2,5-DHMB PND28 M 907.0 ± 530.3 5,496.7 ± 1102.9 11,860.0 ± 1546.5 20,300.0 ± 2805.9 F 528.0 ± 410.0 4,900.0 ± 680.1 11,580.0 ± 1339.2 19,660.0 ± 7940.6 PND56 M 20.0 ± 0.0 7,924.0 ± 959.3 25,740.0 ± 1202.7d 48,400.0 ± 9493.2 F 20.0 ± 0.0 9,484.0 ± 1325.2d 29,640.0 ± 2903.9d 36,725.0 ± 2101.7 Analyte . Age . Sex . Dose (ppm) . 0 (ng/mL) Mean ± SE . 3,000 (ng/mL) Mean ± SE . 10,000 (ng/mL) Mean ± SE . 30,000 (ng/mL) Mean ± SE . HMB PND28 M 590.9 ± 321.9a 3,470.0 ± 1,051.9b 8,822.0 ± 2,781.5b 16,940.0 ± 5498.9b F 352.0 ± 211.0c 2,080.0 ± 156.9b 7,694.0 ± 2501.4b 16,703.3 ± 6908.5c PND56 M 81.7 ± 2.7b 10,600.0 ± 1495.6b 40,100.0 ± 1499.0b,d 5,7900.0 ± 8494.8a F 79.82 ± 2.42b 2,572.0 ± 369.7b 12,204.0 ± 1584.9b 43,350.0 ± 7468.2b DHB PND28 M 1,147.8 ± 723.9 7,941.0 ± 1868.1a 13,182.0 ± 2498.9b 25,260.0 ± 5044.8b F 776.5 ± 613.5c 5,850.0 ± 542.3b 13,758.0 ± 1823.6b 20,636.7 ± 7998.9c PND56 M 0.8 ± 0.0 21,526.0 ± 4205.2b 40,840.0 ± 6861.7b,d 56,250.0 ± 6121.8b F 20.1 ± 0.0 14,006.0 ± 2701.3b,d 25,460.0 ± 3176.1b,d 48,875.0 ± 6316.8b THB PND28 M 183.0 ± 61.0 1,463.3 ± 141.9 2,614.0 ± 380.7 7,908.0 ± 1394.0 F 166.0 ± 68.0 1,173.3 ± 17.6 3,744.0 ± 292.3 5,740.0 ± 1924.3 PND56 M 40.9 ± 0.0 2,830.0 ± 247.9 8,146.0 ± 737.4d 14,037.5 ± 3006.1 F 40.9 ± 0.0 3,160.0 ± 392.2d 11,696.0 ± 800.4d 17,050.0 ± 2554.2 2,5-DHMB PND28 M 907.0 ± 530.3 5,496.7 ± 1102.9 11,860.0 ± 1546.5 20,300.0 ± 2805.9 F 528.0 ± 410.0 4,900.0 ± 680.1 11,580.0 ± 1339.2 19,660.0 ± 7940.6 PND56 M 20.0 ± 0.0 7,924.0 ± 959.3 25,740.0 ± 1202.7d 48,400.0 ± 9493.2 F 20.0 ± 0.0 9,484.0 ± 1325.2d 29,640.0 ± 2903.9d 36,725.0 ± 2101.7 aSignificantly higher than the free metabolite (P < 0.05). bSignificantly higher than the free metabolite (P < 0.01). cComparison was not available due to having only two non-missing pairs. dSignificantly higher than PND28 (P < 0.05). Open in new tab Figure 4. Open in new tabDownload slide Total HMB (A), DHB (B), THB (C), and 2,5-DHMB (D) concentrations in PND28 and PND56 male and female rat plasma following exposure to 0 (control), 3,000, 30,000 ppm HMB via feed. Figure 4. Open in new tabDownload slide Total HMB (A), DHB (B), THB (C), and 2,5-DHMB (D) concentrations in PND28 and PND56 male and female rat plasma following exposure to 0 (control), 3,000, 30,000 ppm HMB via feed. Discussion HMB use poses increasing concern due to exposure through product application in humans. Due to its highly lipophilic nature, it is known to be rapidly absorbed through skin and capable of bioaccumulation (2). It has been found in human urine and milk (20, 27), and also in fish tissue (41–43). Due to the varying degrees of effects of HMB and its metabolites on estrogenic and anti-androgenic activities (25), there is increasing concern about the potential health effects of in utero/postnatal exposure to HMB. The presence of HMB in urine of humans, premature infants and in human breast milk may indicate placental and lactational transfer (44, 45), due to lack of adverse health effects of HMB in humans following dermal application (22), the significance of its presence in human urine has not been ascertained. There are methods reported in the literature to measure HMB and its metabolites in urine (26) and plasma samples (29, 31, 35, 36, 46) with some limitations such as low sensitivity to measure simultaneous free and total analytes with reproducible results in plasma samples. In this study, we have developed and validated a sensitive and selective LC–MS-MS method to simultaneously quantitate HMB and its metabolites DHB, THB and 2,5-DHMB in HSD rat plasma. The method was applied to PND28 and PND56 male and female rat plasma samples following perinatal dietary exposure to 0, 3,000, 10,000 and 30,000 ppm HMB via feed beginning on GD6. While HMB and its metabolites, THB, DHB, and 2,2′-DHMB were previously detected in HSD rats following oral administration of HMB (29, 31), to the best of our knowledge, 2,5-DHMB was only detected as an in vitro metabolite (25, 34). Interestingly, in this study, while 2,5-DHMB was found in the plasma samples following exposure to HMB, 2,2′-DHMB was absent. To the extent of our knowledge, this is the first study that reports the quantitation of free and total HMB and its metabolites in plasma in rodents following perinatal dietary exposure to HMB. Free DHB concentrations were higher than the free HMB concentrations in all exposure groups, which is most likely due to the rapid metabolism of HMB. DHB was identified as one of the major metabolites of HMB in previous investigations, and is formed via O-dealkylation (31). Also, compared to HMB, the concentration of DHB in plasma has been shown to decrease slowly (31). No significant sex differences were observed for free HMB or DHB concentrations in any of the exposure groups for either PND28 or PND56. In general, free HMB and DHB concentrations were higher in PND56 compared to PND28 in both males and females and the increase was significant only in 10,000 ppm exposure group. This is potentially due to higher direct exposure to HMB due to increased feed consumption on PND56. Total analyte concentrations in our study were ~60–200 times higher than free levels (Table III) suggesting extensive conjugation of HMB and its metabolites. Total analyte concentrations at 10,000 and 30,000 ppm followed the general pattern HMB ≈ DHB ≈ 2,5-DHMB » THB, while at 3,000 ppm DHB > 2,5-DHMB ≈ HMB » THB. The difference between HMB concentrations at higher doses might be due to the saturation of metabolism (decreased metabolic rate due to the saturation of glucoronide and/or sulfate) by the higher concentration of HMB in plasma. It is not surprising to see that THB had the lowest determined concentrations among total analytes at all dose levels, considering THB was identified as a minor metabolite of HMB which requires multiple Phase 1 and 2 metabolism steps to form, and is likely formed via aromatic hydroxylation of DHB (31). Similar to free HMB, total HMB concentrations at PND56 were in general higher than PND28. The significant difference was only in male 10,000 ppm exposure group. Considering no sex difference was observed between male and female for total and free analyte concentrations across all exposure groups, it is surprising the age effect was not observed in females while it was more apparent in males. No observed sex difference between male and female rats in this study is somewhat contradictory with previously reported possible gender differences in metabolism and distribution of HMB (22). Overall, the concentrations of both free and total analytes increased with increasing HMB exposure and were found to be higher than their corresponding controls in PND28 and PND56 males and females. Previously, Nakamura et al. (46) have also reported increasing free HMB and DHB concentrations with increasing dose in plasma samples from HSD dams at PND23 following dietary exposure to 0 (control), 1,000, 3,000, 10,000, 25,000 and 50,000 ppm HMB from GD6 to PND23, while no free THB or 2,2-DHMB were detected in the same samples. While mean free HMB was found to be approximately dose proportional, free DHB concentrations only increased ~1.5–2-fold with the 3-fold increase in exposure concentration (Table II). A similar trend was observed with total HMB increasing proportionally to the exposure concentration, while total DHB, THB and 2,5-DHMB concentrations increased less than proportionally (Table III). To address the human relevance of this data, we compared our data to the data reported in human plasma following a repeated dermal application of HMB (20 g/m2) as a cream in postmenopausal women (ages 54–86) and young men (ages 3–29) for 4 days (47). In this study, mean free HMB plasma concentrations one hr after exposure were 60 (range: 7–236) and 92 (range: 16–390) ng/mL and the 24 h time point were 47 (range: 0–295) and 41 (range: 8–703) ng/mL in women and men, respectively. We compared the free HMB plasma level ratio and external dose based on body weight (kg)/surface area (m2) ratio between our study and the human dermal study (Table IV). Based on the comparisons, our exposure concentrations and free HMB plasma concentrations were within ~ 0.1–4.0-fold to the human dermal study (47) demonstrating the similarities between the two studies. Table IV. Comparison of animal data to human data based on free HMB plasma levels and external dose Rodent feed exposure concentration (mg HMB/kg feed) . Mean feed consumption (g feed/kg bwt/day) . Mean estimated HMB consumption (mg/kg)a . Estimated doses in (g/m2)b . Rat to human dose ratioc . Rat to human Free HMB plasma ratiod . M . F . M . F . M . F . M . F . M . F . 3,000 95.1 91.0 285.2 273.0 1.7 1.64 0.09 0.08 0.60 0.35 10,000 91.1 112.8 910.9 1,128.2 5.5 6.7 0.27 0.34 2.05 1.64 30,000e 135.7 203.0 4,070.4 6,088.6 24.4 36.5 1.22 1.83 3.65 3.08 Rodent feed exposure concentration (mg HMB/kg feed) . Mean feed consumption (g feed/kg bwt/day) . Mean estimated HMB consumption (mg/kg)a . Estimated doses in (g/m2)b . Rat to human dose ratioc . Rat to human Free HMB plasma ratiod . M . F . M . F . M . F . M . F . M . F . 3,000 95.1 91.0 285.2 273.0 1.7 1.64 0.09 0.08 0.60 0.35 10,000 91.1 112.8 910.9 1,128.2 5.5 6.7 0.27 0.34 2.05 1.64 30,000e 135.7 203.0 4,070.4 6,088.6 24.4 36.5 1.22 1.83 3.65 3.08 aEstimated HMB consumption (mg HMB /kg body weight/day = mean feed consumption (g feed/kg body weight × 1 kg/1,000 g) × exposure concentration (mg HMB/kg feed)). bCorresponding doses in g/m2 were calculated by the following equation, g HMB/m2 = Mean estimated feed consumption (mg/kg body weight) ∗ rat Km factor (6) (kg body weight/m2) × (1 g/1,000 mg) (48). cRat to human ratios calculated by the dividing corresponding rat doses (g/m2) to human study dose (20 g/m2). dRat to human ratios of free HMB plasma concentrations were calculated by diving mean plasma concentration values of male and female Sprague Dawley rats at PND56 (Table II) by mean free HMB plasma values in humans 1 h after HMB exposure (47). eThe reason for observed higher feed consumption in 30,000 ppm group compared to the other lower exposure groups is unknown. M, male; F, female. Open in new tab Table IV. Comparison of animal data to human data based on free HMB plasma levels and external dose Rodent feed exposure concentration (mg HMB/kg feed) . Mean feed consumption (g feed/kg bwt/day) . Mean estimated HMB consumption (mg/kg)a . Estimated doses in (g/m2)b . Rat to human dose ratioc . Rat to human Free HMB plasma ratiod . M . F . M . F . M . F . M . F . M . F . 3,000 95.1 91.0 285.2 273.0 1.7 1.64 0.09 0.08 0.60 0.35 10,000 91.1 112.8 910.9 1,128.2 5.5 6.7 0.27 0.34 2.05 1.64 30,000e 135.7 203.0 4,070.4 6,088.6 24.4 36.5 1.22 1.83 3.65 3.08 Rodent feed exposure concentration (mg HMB/kg feed) . Mean feed consumption (g feed/kg bwt/day) . Mean estimated HMB consumption (mg/kg)a . Estimated doses in (g/m2)b . Rat to human dose ratioc . Rat to human Free HMB plasma ratiod . M . F . M . F . M . F . M . F . M . F . 3,000 95.1 91.0 285.2 273.0 1.7 1.64 0.09 0.08 0.60 0.35 10,000 91.1 112.8 910.9 1,128.2 5.5 6.7 0.27 0.34 2.05 1.64 30,000e 135.7 203.0 4,070.4 6,088.6 24.4 36.5 1.22 1.83 3.65 3.08 aEstimated HMB consumption (mg HMB /kg body weight/day = mean feed consumption (g feed/kg body weight × 1 kg/1,000 g) × exposure concentration (mg HMB/kg feed)). bCorresponding doses in g/m2 were calculated by the following equation, g HMB/m2 = Mean estimated feed consumption (mg/kg body weight) ∗ rat Km factor (6) (kg body weight/m2) × (1 g/1,000 mg) (48). cRat to human ratios calculated by the dividing corresponding rat doses (g/m2) to human study dose (20 g/m2). dRat to human ratios of free HMB plasma concentrations were calculated by diving mean plasma concentration values of male and female Sprague Dawley rats at PND56 (Table II) by mean free HMB plasma values in humans 1 h after HMB exposure (47). eThe reason for observed higher feed consumption in 30,000 ppm group compared to the other lower exposure groups is unknown. M, male; F, female. Open in new tab In our study, we detected low levels of free and total analytes in most of the control samples. Free HMB and DHB concentrations in control groups ranged between 1.38–3.97 and 3.11–6.84 ng/mL, respectively (Tables II and III), however, it is unclear whether this observed levels were due to low levels of environmental exposure and/or post study background levels during the conduct of the assay. In order to investigate this we compared the ratio of HMB to DHB in controls and the lowest exposure group of 3,000 ppm. The ratio of HMB/DHB in exposed group was much higher than the control group. Suggesting that the control animals were not exposed to HMB, rather the levels observed in the control animals were likely due to contamination of the samples during necropsy and/or sample analysis. A recent study also reported HMB levels in plasma from control HSD dams at PND23 following dietary exposure (0, 1,000, 3,000, 10,000, 25,000 and 50,000 ppm) to HMB in feed from GD6 to PND23 with levels reported at 11.8 ± 7.0 ng/mL (mean ± S.E.) but no free DHB, THB or 2,2-DHMB were detected in any of the samples analyzed (46). Conclusions Free HMB and DHB, and total HMB, DHB, THB and 2,5-DHMB were simultaneously quantitated in rat plasma following extraction for free and acid hydrolysis for total analytes. All analytes were precisely and accurately quantitated over the ranges of 15–150 ng/mL for free HMB and DHB, 200–2,000 ng/mL for total HMB and THB, and 40–400 ng/mL for total DHMB and DHB. The validated method was successfully applied to the analysis of these analytes in HSD rat plasma following administration of 0, 3,000, 10,000 and 30,000 ppm HMB via feed. Additionally, plasma samples as high as 60 μg/mL for free HMB and DHB and 175 μg/mL for total HMB, DHB, THB and 2,5-DHMB, were successfully diluted into the validated concentration range and analyzed. While all the total analytes were quantified as total in all plasma samples, the only free analytes detected in plasma samples were HMB and DHB. Total concentrations were considerably higher than free concentrations, highlighting extensive conjugation. Free DHB concentrations were greater than free HMB concentrations. Overall, total analyte concentrations in PND56 plasma were found to be higher than in PND28 plasma samples. No significant differences were found between male and female PND28 or PND56 samples for free and total analyte concentrations. There was a dose and time dependent increase in total analyte concentrations which were not sex-dependent. 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Google Scholar Crossref Search ADS WorldCat Published by Oxford University Press 2017. This work is written by (a) US Government employee(s) and is in the public domain in the US. Published by Oxford University Press 2017.
TI - Simultaneous Quantitation of 2-Hydroxy-4-Methoxybenzophenone, a Sunscreen Ingredient, and its Metabolites in Harlan Sprague Dawley Rat Plasma Following Perinatal Dietary Exposure
JF - Journal of Analytical Toxicology
DO - 10.1093/jat/bkx070
DA - 2017-11-01
UR - https://www.deepdyve.com/lp/oxford-university-press/simultaneous-quantitation-of-2-hydroxy-4-methoxybenzophenone-a-uyOJjZkldb
SP - 744
EP - 754
VL - 41
IS - 9
DP - DeepDyve
ER -