Abstract
A rapid two minute liquid chromatography tandem mass spectrometry (LC-MS/MS) method operating in multiple reaction ion monitoring mode was developed and validated that allows for the characterization and simultaneous quantification of eleven phytoestrogen metabolites with mass transitions m/z 241/119 (equol), 253/132(daidzein), 255/149 (dihydrodaidzein), 257/108 (O-desmethylangolesin), 269/133 (genistein), 283/184 (glycitein), 267/191 (formononetin), 289/109 (biochanin A), 267/91 (coumestrol), enterodiol (301/253), and enterolactone (297/253). The method was demonstrated to be specific and sensitive, and a linear response for each phytoestrogen was observed over a range of 1–5,000 ng/mL in human serum with the exception of dihydrodaidzein, whose lower limit of quantification was 2 ng/mL. The separation was carried out on a Synergi Polar-RP 2.5 micron (50 × 2.0 mm, i.d.) column at 50°C with water and acetonitrile (both containing 10 mM ammonium acetate) as the mobile phase under gradient conditions at a flow rate of 0.75 mL/min. This LC-MS/MS method is very useful for high throughput analysis of phytoestrogens and proved to be simple, sensitive, reproducible, and reliable.
Keywords: LC-MS/MS, phytoestrogens, serum, analysis
INTRODUCTION
Phytoestrogens are structurally diverse diphenolic compounds that are present in plants and may impact human health, at least in part, by virtue of their estrogenic effects. Isoflavones, coumestanes and lignans are the major biologically active phytoestrogens. Most phytoestrogens bind to estrogen receptors (α and β) and may function as estrogen agonists and antagonists. The potential role(s) of dietary phytoestrogens in the prevention of chronic diseases (e.g., cancer, heart disease, neurodegeneration and osteoporosis) is of substantial interest (1–8). Epidemiological evidence suggests that consumption of phytoestrogens is associated with a lower risk of breast cancer (9,10) and animal studies have demonstrated that early life exposure to the soy isoflavones reduces the number of mammary tumors induced by carcinogens (11,12). On the other hand, there are reports suggesting that phytoestrogens may increase the growth of pre-existing breast cancer (13,14). As for steroid hormones, most phytoestrogens have an enterohepatic circulation and are metabolized by phase II enzymes to glucuronide and sulfate conjugates.
We initially developed a specific, quantitative LC-MS/MS method for these compounds in 1996 (15) and subsequently improved it in 2004 (16). However, research on phytoestrogens requires an ever-increasing number of samples to be analyzed. Thus, analytical methods that are sensitive, reproducible and high throughput are needed to evaluate the beneficial or adverse effects of phytoestrogens in numerous pre-clinical and clinical studies. There are several reports on analytical techniques used in quantification of phytoestrogens in biological samples (17–19). HPLC with UV or coulometric array detection has been used by several investigators for quantification of phytoestrogens (20–22). However, these methods suffer from poor sensitivity and selectivity (23). Starkey et al. (2002) used capillary electrochromatography to determine trace isoflavone phytoestrogens in biological samples (24). Gas chromatography-mass spectrometry (GC-MS) has been used as a sensitive analytical technique for phytoestrogen analysis (25–28). However, the large sample volume, multi-step sample preparation procedures and requirement of derivatization prior to analysis for polar and involatile compounds limit the usefulness of GC-MS analysis. LC-MS/MS has become the method of choice due to its high sensitivity, selectivity and easy sample preparation (29– 31). Grace et al. (2007) reported quantification of phytoestrogens in human urine and serum using LC-MS/MS (31). However, the method involves a ten minute chromatographic run time for analysis of nine phytoestrogens. Another LC-MS/MS method for the determination of urinary phytoestrogens has been reported (32). However, the method suffers from a relatively long chromatographic run time (9 min) and small number of phytoestrogens.
The aim of this study was to develop and validate a multi-target, high-throughput LC-MS/MS method for the quantification of phytoestrogens in serum. We now describe a rapid two minute LC-MS/MS method for simultaneous quantification of eleven phytoestrogens in serum (Fig. 1), thus making the assay suitable for high-throughput analysis required for analysis of large number of samples in phytoestrogen research.
Fig. 1.
Chemical structures of standard phytoestrogens.
EXPERIMENTAL
Chemicals
Genistein, daidzein, dihydrodaidzein (DHD), equol, O-desmethylangolesin (O-DMA), glycitein, biochanin A, coumestrol, enterodiol, enterolactone, and chrysin were purchased from LC-laboratories (Woburn, MA). All HPLC solvents and reagents were purchased from Fisher Scientific Co. (Norcross, GA) and were of HPLC grade. Phenolphthalein β-glucuronide, 4-methylumbelliferone sulfate and β-Glucuronidase/sulfatase from Helix pomatia was purchased from Aldrich-Sigma Chemical Co. (St, Louis, MO).
Sample preparation
We previously showed that the average recovery of isoflavones as a group using diethyl ether was ~90%, whereas their recoveries using Sep-pak C18 cartridge was only 82% (15). We therefore, used liquid-liquid extraction of phytoestrogens in serum with diethyl ether for sample preparation. Blank serum samples were obtained from Millipore Corporation (Temecula, CA). Stock solutions of individual phytoestrogens were prepared in DMSO and then diluted with methanol-water (80:20 v/v) to obtain appropriate working solution containing all phytoestrogens and the internal standard (IS, chrysin; 200 ng/mL). For calibration standards and quality control (QC) samples, each blank serum sample (200 µL) was spiked with appropriate concentration of working solution of phytoestrogens. Since hydrolysis is an important step that converts the conjugated forms into the aglycone forms of phytoestrogens, enzymatic hydrolysis of the phytoestrogen glucuronides and sulfates was used for the total phytoestrogen determination in biological samples. We, therefore, followed the extraction procedure that mimics the extraction of conjugated metabolites. Spiked serum samples were hydrolyzed by incubating with β-glucuronidase/sulfatase at 37 °C overnight and extracted into diethyl ether using our reported method (15). Phenolphthalein β-glucuronide, 4-methylumbelliferone sulfate were added to monitor completeness of hydrolysis with β-glucuronidase/sulfatase. After concentrating samples to dryness, they were dissolved in methanol-water (80:20, v/v) prior to LC-MS/MS analysis.
Validation study
The analytical method was validated to demonstrate the specificity, recovery, lower limit of quantification (LLOQ), accuracy, and precision of measurements. Specificity was established by the lack of interference peaks at the retention time for the internal standards and phytoestrogens. Linearity was tested at 7 levels of concentrations covering a range of 1–5000 ng/mL. The regression parameters of slope, intercept and correlation coefficient were calculated by linear least-square regression (1/x2 weighting).
The recovery of the method was determined by comparing the peak areas obtained from the blank serum samples spiked prior to extraction with the peak areas obtained from spiked post-extraction serum samples. The matrix effect was evaluated for all analytes and the internal standard. For this, blank serum samples were processed according to the liquid-liquid extraction method described above and then spiked with phytoestrogens and IS at the final concentration after extraction. The matrix effect for each phytoestrogens was calculated by comparison of mean peak area (n = 6) obtained for blank serum samples spiked with phytoestrogens and IS after extraction and peak area of standards in methanol-water (80:20, v/v) at concentrations 50, 500 and 2000 ng/mL. The matrix effect results obtained in this study were calculated as follows: Matrix effect = [1−Y/X] × 100%, where X and Y represent the mean peak areas of phytoestrogen standards in methanol-water (80:20, v/v) and spiked post extraction at corresponding concentrations, respectively.
The accuracy and precision (presented as %CV) of this analytical method were determined using quality control (QC) samples in 4–6 replicates of 50, 500, and 2000 ng/mL of phytoestrogens in serum. Accuracy was determined by comparing the calculated concentration using calibration curves to nominal concentration. The limit of quantification (LOQ) was defined as precision and accuracy within 20%. The LLOQ was defined as the smallest amount of the analyte that could be measured in a sample with sufficient precision and accuracy (within 20% for both parameters) and was chosen as the lowest concentration on the calibration curve.
Liquid chromatography-mass spectrometry
LC-MS/MS analyses of serum samples were performed using a system consisting of a Shimadzu Prominence HPLC with a refrigerated auto sampler (Shimadzu Scientific Instruments, Inc. Columbia, MD), and an API 4000 (Applied Biosystems/MDS Sciex, Concord, Ontario, Canada) triple quadrupole mass spectrometer. Chromatography was performed on a Synergi 2.5 micron Polar-RP 100A column (50 mm × 2.0 mm i.d. column with a 5 mm × 2.0 mm i.d. guard) at a flow rate of 0.75 mL/min. The mobile phase consisted of (A) 10 mM ammonium acetate and (B) acetonitrile containing 10 mM ammonium acetate, using a gradient elution of 25–70%B at 0.75 min and return to 25%B at 1.0 min. The temperature of the column oven was 50°C. The column effluent was introduced into the mass spectrometer using an electrospray ionization interface operating in the negative ion mode. The source temperature was set at 400°C and ion spray voltage of 4 kV. Nitrogen was used as nebulizer, secondary and curtain gas. The tandem mass spectrometer was tuned in the multiple reaction monitoring mode to monitor mass transitions m/z 241/119 (equol), 253/132(daidzein), 255/149 (dihydrodaidzein), 257/108 (O-desmethylangolesin), 269/133 (genistein), 283/184 (glycitein), 267/191 (formononetin), 289/109 (biochanin A), 267/91 (coumestrol), 301/253 (enterodiol), 297/253 (enterolactone), internal standards 175/119 (4-methylumbelliferone), 317/93 (phenolphthalein) and 253/143 (chrysin). The optimized MS/MS operating parameters used in this study are listed in Table 1. Sample injections of 10 µL were carried out and the autosampler temperature was set at 4°C.
Table 1.
MS/MS parameters optimized for phytoestrogens and internal standards
| Analyte | Q1/Q3 | Dwell (msec) | DPh (V) |
CE (eV) |
CXP (V) |
|---|---|---|---|---|---|
| Equol | 314/119 | 50 | −65 | −30 | −5 |
| Daidzein | 253/132 | 50 | −65 | −55 | −10 |
| Dihydrodaizein | 255/149 | 50 | −50 | −30 | −9 |
| O-DMA | 257/108 | 50 | −70 | −40 | −5 |
| Genistein | 269/133 | 50 | −75 | −40 | −5 |
| Glycitein | 283/184 | 50 | −65 | −45 | −5 |
| Formononetin | 267/251 | 50 | −75 | −35 | −5 |
| Coumestrol | 267/91 | 50 | −50 | −50 | −2 |
| Biochanin A | 283/268 | 50 | −70 | −30 | −5 |
| Enterolactone | 297/253 | 50 | −80 | −30 | −10 |
| Enterodiol | 301/253 | 50 | −70 | −30 | −9 |
| Phenophthalein | 317/93 | 50 | −50 | −20 | −5 |
| 4-MU | 175/119 | 50 | −50 | −38 | −4 |
| Chrysin | 253/143 | 50 | −50 | −50 | −5 |
DP = Declustering potential
CE = Collision energy
CXP = Cell exit potential
RESULTS AND DISCUSSION
Chromatography and mass spectrometry
Fast chromatographic separation of eleven phytoestrogens with acceptable resolution for LC-MS/MS was a major challenge. In order to optimize the separation and detection of phytoestrogens, variables such as column type, column temperature, gradient, and flow rate were investigated. Our original LC-MS/MS method involved a gradient analysis that had a chromatographic run time of 15 min per sample. Raising the column temperature to 40°C, increasing the flow rate from 0.2 mL/min to 0.4 mL/min and the shortening the column length from 150 mm to 30 mm, reduced the run time to 5 min. We subsequently found that the phytoestrogens were stable at 50°C in the mobile phase, a temperature that allowed the flow rate to be further increased to 0.75 ml/min. Phenyl hexyl columns with different particle sizes were tested for better resolution. By decreasing the column particle size to 2.5 µm while extending the column length to 50 mm, an acceptable chromatographic separation was obtained with a run time of 2 min (Fig. 2). Since the injection takes about 0.2 min for each consecutive injection, a total of 27 samples can be analyzed using this method per hour. Table 2 summarizes experimental parameters for detection of phytoestrogens in 15, 5 and 2 min LC-MS/MS runs.
Fig. 2.
MRM chromatogram showing separation of eleven phytoestrogens using a 2 min run time.
Table 2.
Comparison of LC-MS/MS methods with 15, 5 and 2 min run times for phytoestrogen analysis
| 15 min method | 5 min method | 2 min method |
|---|---|---|
| Mobile phase | ||
| 10 mM NH4OH (solvent A) | 10 mM NH4OH (solvent A) | 10 mM NH4OH (solvent A) |
| MeCN with 10 mM | MeCN with 10 mM | MeCN with 10 mM |
| NH4OH (solvent B) | NH4OH (solvent B) | NH4OH (solvent B) |
| Column | ||
| Phenomenex Phenyl-Hexyl | Phenomenex Phenyl-Hexyl | Phenomenex polar-RP |
| 2.0×150 mm with guard | 2.0×30 with guard | 2.0×50 mm guard |
| Particle size 3 µ | Particle size 3 µ | Particle size 2.5 µ |
| Column temperature | ||
| Ambient | 40 °C | 50 °C |
| Flow rate | ||
| 0.2 mL | 0.4 mL | 0.75mL |
| HPLC pump | ||
| Shimadzu LC-10 AD | Shimadzu LC-10 AD | Shimadzu LC-20 AD |
| Gradient | ||
| 0 min = 20%B | 0 min = 25%B | 0 min = 25%B |
| 10 min = 70%B | 2 min = 80%B | 0.75 min = 70%B |
| 11 min = 20%B | 2.5 min = 25%B | 1.0 min = 25%B |
| 15 min = stop | 5 min = stop | 2 min = stop |
Deprotonated molecular ions [M−H]− of all phytoestrogen standards were induced to fragment in the collision cell, and after optimization, the most abundant product ions among the resulting product ion spectra were chosen for quantitative MRM of all phytoestrogens (Table 1).
Method validation
The methods were validated in accordance with the FDA guidance for bioanalytical method validation and also based on the paper of Shah et al. (33,34). A full validation was performed for rat serum based on the following criteria:
Linearity and dynamic range
The most desirable analytical method typically combines an efficient chromatographic separation with short run time and required sensitivity. An epidemiological study shows that the plasma concentrations of phytoestrogens such as daidzein (20 µg/L) and genistein (40 µg/L) vary 5–50 times among participants (35). It is therefore, desirable to develop a LC-MS/MS assay method with a wide dynamic range. This assay was linear for each of the analytes over a range of 1–5000 ng/mL in serum with correlation coefficients > 0.99, except for dihydrodaizein, whose lower limit of quantification (LLOQ) was 2 ng/mL. At the LLOQ of phytoestrogens accuracy ranged from 95.53 to 114.83% with precision in the range of 3.08 to 13.49%. At all concentrations, accuracy ranged from 87.53 to 114.83% and precision ranged from 0.83% to 14.85%. These results indicated a linear relationship between the peak areas and concentrations of all standard phytoestrogens in this developed assay.
The accuracy and precision (presented as %CV) of this analytical method were determined using QC samples at concentrations 50, 500 and 2000 ng/mL in five-six replicates. The performance characteristics of the method were established by validation procedures employing assays with standard solutions, sample blanks and spiked samples. The intra- and inter-batch precision and accuracy were found to be well within acceptable limits as described above (Table 3). At the lowest quality control (50 ng/mL), the intra- day precisions for the phytoestrogens were less than 15% and the accuracies were between 80–114.66 %. The inter-day precision all concentrations ranged from 2.82 to 8.59%. This ultra fast method is very useful for high-throughput analysis of phytoestrogens and proved to be reproducible, reliable and the results were comparable to those obtained by regular flow LC/MS/MS analysis.
Table 3.
Precision and accuracy of quality control samples
| Analyte | Nominal concentration (ng/mL) |
Accuracy (%) | Precision (%CV) | |||||
|---|---|---|---|---|---|---|---|---|
| Day 1 | Day 2 | Day 3 | Day 1 | Day 2 | Day 3 | Inter-day | ||
| Equol | 50 | 100.42 | 90.13 | 96.60 | 2.01 | 4.33 | 5.11 | 3.74 |
| 500 | 103.30 | 99.85 | 114.66 | 2.31 | 5.61 | 1.93 | 2.97 | |
| 2,000 | 97.60 | 89.90 | 103.96 | 6.11 | 10.61 | 10.13 | 8.34 | |
| Daidzein | 50 | 99.98 | 102.73 | 94.04 | 4.35 | 6.44 | 8.23 | 6.62 |
| 500 | 101.48 | 98.31 | 97.73 | 3.14 | 5.44 | 7.42 | 5.38 | |
| 2,000 | 92.50 | 87.41 | 86.03 | 2.88 | 3.61 | 3.96 | 3.58 | |
| Dihydrodaidzein | 50 | 103.00 | 100.15 | 101.66 | 3.94 | 1.43 | 4.99 | 3.63 |
| 500 | 103.79 | 95.20 | 106.00 | 3.96 | 6.44 | 3.35 | 4.34 | |
| 2,000 | 91.70 | 90.40 | 96.33 | 1.68 | 5.80 | 6.60 | 2.82 | |
| O-DMA | 50 | 104.00 | 93.72 | 96.51 | 5.16 | 4.71 | 5.80 | 5.32 |
| 500 | 105.67 | 93.78 | 102.33 | 3.22 | 9.42 | 5.54 | 5.84 | |
| 2,000 | 101.20 | 93.57 | 100.93 | 5.53 | 5.37 | 6.53 | 3.63 | |
| Genistein | 50 | 107.66 | 106.83 | 99.08 | 3.97 | 3.37 | 6.65 | 4.86 |
| 500 | 97.50 | 88.90 | 91.36 | 5.40 | 3.61 | 5.60 | 4.96 | |
| 2,000 | 95.13 | 92.28 | 93.38 | 2.63 | 3.97 | 4.17 | 3.59 | |
| Glycitein | 50 | 97.00 | 80.00 | 92.13 | 11.64 | 7.77 | 7.52 | 8.59 |
| 500 | 100.66 | 91.96 | 99.96 | 6.76 | 5.09 | 5.24 | 5.58 | |
| 2,000 | 96.20 | 92.70 | 100.21 | 2.34 | 5.87 | 5.19 | 4.29 | |
| Formononetin | 50 | 95.43 | 98.65 | 98.50 | 4.28 | 7.69 | 4.93 | 5.59 |
| 500 | 101.53 | 96.60 | 108.66 | 4.95 | 6.09 | 2.99 | 4.35 | |
| 2,000 | 92.72 | 94.08 | 101.90 | 2.09 | 7.80 | 2.26 | 3.78 | |
| Coumestrol | 50 | 92.97 | 89.24 | 99.42 | 8.66 | 9.15 | 6.69 | 7.66 |
| 500 | 93.86 | 93.95 | 96.03 | 9.05 | 7.78 | 5.34 | 7.26 | |
| 2,000 | 100.45 | 89.95 | 84.12 | 7.83 | 7.34 | 3.66 | 6.95 | |
| Biochanin A | 50 | 104.23 | 103.13 | 107.50 | 6.82 | 3.88 | 3.26 | 4.48 |
| 500 | 89.95 | 94.01 | 98.41 | 4.46 | 6.43 | 5.24 | 5.15 | |
| 2,000 | 98.88 | 95.00 | 93.68 | 2.91 | 3.10 | 5.47 | 3.90 | |
| Enterodiol | 50 | 93.35 | 88.63 | 96.95 | 9.22 | 7.46 | 5.36 | 7.11 |
| 500 | 104.95 | 98.95 | 112.16 | 7.81 | 4.10 | 4.12 | 4.98 | |
| 2,000 | 96.50 | 87.40 | 94.73 | 3.98 | 6.05 | 7.94 | 5.86 | |
| Enterolactone | 50 | 104.15 | 102.20 | 108.83 | 9.23 | 5.71 | 2.50 | 5.57 |
| 500 | 103.30 | 92.26 | 104.00 | 5.43 | 8.33 | 1.98 | 5.03 | |
| 2,000 | 84.68 | 83.33 | 91.90 | 1.52 | 0.35 | 7.56 | 3.09 | |
Specificity and selectivity
Representative MRM chromatograms of extracted blank serum and blank serum spiked with phytoestrogens (50 ng/mL) and internal standards (IS) are shown in Fig. 3. The LC-MS/MS method demonstrated high specificity because only ions derived from phytoestrogens and the internal standards were observed, indicating that there was no endogenous substance in serum interfering in the analysis of phytoestrogens.
Fig. 3.
Representative MRM chromatograms of serum spiked with phytoestrogens (50 ng/mL) (A); internal standards 4-MU, phenolpthalein, and chrysin (B).
Carry over
Carry-over is one of the most commonly encountered problems in the quantification of metabolites in biological samples by LC/MS/MS. As can be seen in Fig. 4, when a methanol blank was injected right after the highest level of phytoestrogens spiked serum sample (5000 ng/mL), little or no detectable carryover was observed on the column. Note that full-scale for the methanol blank was 270 times smaller than for the phytoestrogen standards. Since the peak intensities were also much lower, the carryover was estimated to be less than 0.2%.
Fig. 4.
Representative MRM chromatograms for phytoestrogens in plasma (5000 ng/mL) (A); 10 µL methanol blank (B).
Matrix effects and recovery
Evaluating the matrix effect (ion suppression or enhancement) has become an integral part of method development and validation (36, 37). Blank serum samples were extracted, then spiked and analyzed for potential interferences by endogenous matrix components. The matrix effects from serum on each phytoestrogen analytes are listed in Table 4. At 5, 500 and 2000 ng/mL concentration levels, the analytes had matrix effects within the range of −13 to 34%. These results indicated that there were minor to moderate matrix effects for phytoestrogens in serum (38).
Table 4.
Calculated % matrix effects of serum on the peak area response of phytoestrogens and internal standard
| Analyte | Concentration (ng/mL) | Matrix effect (%) |
|---|---|---|
| Equol | 50 | 2.78 |
| 500 | 18.28 | |
| 2000 | 4.55 | |
| Daidzein | 50 | 16.38 |
| 500 | 9.08 | |
| 2000 | 2.51 | |
| Dihydrodaidzein | 50 | 13.22 |
| 500 | 15.21 | |
| 2000 | 3.00 | |
| O-DMA | 50 | 1.22 |
| 500 | 14.52 | |
| 2000 | −0.41 | |
| Genistein | 50 | 30.99 |
| 500 | 28.51 | |
| 2000 | 3.84 | |
| Glycitein | 50 | 21.01 |
| 500 | 25.33 | |
| 2000 | −0.55 | |
| Formononetin | 50 | 34.95 |
| 500 | 28.18 | |
| 2000 | 4.50 | |
| Coumestrol | 50 | 31.90 |
| 500 | 20.57 | |
| 2000 | −2.42 | |
| Biochanin A | 50 | 23.82 |
| 500 | 14.85 | |
| 2000 | 20.62 | |
| Enterodiol | 50 | 15.15 |
| 500 | 9.82 | |
| 2000 | −12.03 | |
| Enterolactone | 50 | −13.66 |
| 500 | 1.65 | |
| 2000 | −12.29 | |
| Chrysin (IS) | 200 | 25.09% |
Recovery studies of phytoestrogens were performed at concentrations of 50, 500 and 5000 ng/mL by comparing the peak areas of the extracted plasma samples with those of post spiked samples. Overall mean extraction recoveries at these concentrations for equol, daidzein, dihydrodaidzein, O-DMA, genistein, glycitein, formononetin, coumestrol, biochanin A, enterodiol, enterolactone, were 83.20%, 82.05%, 88.43%, 75.89%, 57.48%, 86.20%, 85.15%, 77.09%, 65.59%, 86.66% and 81.43%, respectively.
Stability
The stability of QC samples was investigated thoroughly to evaluate their stability in serum samples under different conditions (autosampler and long term storage stability). The stability data are summarized in Table 5. Phytoestrogens are stable in serum for at least 72 h of storage in the autosampler. The autosampler stability was evaluated at 4°C over 72 h and precision of QC samples ranged from 1.88 to 14.44, indicating that they are stable for at least 72 h in human serum when stored at 4°C.
Table 5.
Stability of quality control samples
| Compound | Nominal Concentration (ng/mL) |
Mean measured concentration (ng/mL) | |
|---|---|---|---|
| autosampler at 4 °C, 72h | long storage −20 °C, 2 months | ||
| Equol | 50 | 43.35 ± 2.50 | 45.68 ± 3.98 |
| 500 | 487.80 ± 9.20 | 475.66 ± 30.16 | |
| 2000 | 1793.33 ± 67.42 | 1921.66 ± 94.74 | |
| Daidzein | 50 | 47.03 ± 2.50 | 50.83 ± 1.87 |
| 500 | 534.20 ± 21.05 | 491.66 ± 7.17 | |
| 2000 | 1848.33 ± 72.77 | 1861.66 ± 71.67 | |
| Dihydrodaidzein | 50 | 45.55 ± 1.97 | 47.52 ± 5.23 |
| 500 | 485.83 ± 26.35 | 219.20 ± 15.90 | |
| 2000 | 1738.33 ± 85.18 | 828.50 ± 27.01 | |
| O-DMA | 50 | 48.31 ± 3.75 | 54.80 ± 5.67 |
| 500 | 469.16 ± 24.01 | 534.66 ± 28.57 | |
| 2000 | 1861.66 ± 114.61 | 2151.66 ± 110.89 | |
| Genistein | 50 | 50.90 ± 3.19 | 51.16 ± 3.34 |
| 500 | 487.33 ± 33.15 | 497.33 ± 37.59 | |
| 2000 | 1875.00 ± 116.40 | 2190.00 ± 11.83 | |
| Glycitein | 50 | 44.31 ± 2.44 | 40.15 ± 1.98 |
| 500 | 481.00 ± 39.11 | 489.50 ± 28.26 | |
| 2000 | 1886.66 ± 87.10 | 2045.00 ± 191.91 | |
| Formononetin | 50 | 47.36 ± 4.16 | 47.58 ± 3.22 |
| 500 | 512.33 ± 26.41 | 507.66 ± 27.82 | |
| 2000 | 2018.33 ± 106.09 | 1925.00 ± 167.06 | |
| Coumestrol | 50 | 46.26 ± 6.68 | 56.80 ± 2.37 |
| 500 | 549.33 ± 36.74 | 498.00 ± 26.1 | |
| 2000 | 2120.00 ± 104.30 | 1905.00 ± 128.17 | |
| Biochanin A | 50 | 52.47 ± 2.27 | 56.10 ± 1.49 |
| 500 | 444.00 ± 29.81 | 523.00 ± 23.34 | |
| 2000 | 1893.33 ± 202.06 | 2130.00 ± 88.31 | |
| Enterodiol | 50 | 44.96 ± 3.45 | 46.84 ± 2.47 |
| 500 | 488.16 ± 13.04 | 489.83 ± 20.79 | |
| 2000 | 1906.66 ± 68.89 | 1963.33 ± 119.27 | |
| Enterolactone | 50 | 52.20 ± 3.27 | 48.73 ± 6.55 |
| 500 | 495.50 ± 16.92 | 484.66 ± 34.44 | |
| 2000 | 1801.66 ± 75.70 | 1678.00 ± 66.55 | |
Repeated freeze and thawing of plasma samples spiked with phytoestrogens did not affect their stability. The longer term (over 2 months) stability of phytoestrogens in human serum was evaluated by analyzing frozen QC samples in six replicates. All compounds showed acceptable range of concentration except for dihydrodaidzein at concentrations 500 and 2000 ng/mL (Table 5). It is possible that dihydrodaidzein may decompose when stored over two months in human serum.
Analysis of phytoestrogens in serum samples
The method described was successfully applied for quantification of phytoestrogens in serum samples collected from a study of dietary and serum phytoestrogens and women’s health conditions in midlife (Table 6). The samples were analyzed in duplicate. MRM chromatograms showing detection of various phytoestrogens in a serum samples are shown in Fig. 5. The most abundant phytoestrogens were dihydrodaidzein (31.60 ng/mL), followed by daidzein (27.43 ng/mL) and genistein (26.83 ng/mL). No coumestrol was detected from the sample. It indicates that isoflavones daidzein and genistein are the major components in their diet. These data show the applicability of this assay in quantification of phytoestrogens in large population based studies.
Table 6.
Mean concentration, standard deviation and %CV for phytoestrogen levels in human serum samples (ng/mL)
| Equol | daidzein | DHD | O-DMA | Genistein | Glycitein | Formononetin | Biochanin A | Enterodiol | Enterolactone | |
|---|---|---|---|---|---|---|---|---|---|---|
| Mean | 24.17 | 27.43 | 31.61 | 19.24 | 26.83 | 12.49 | 0.26 | 1.05 | 5.26 | 9.99 |
| STDV | 0.72 | 0.72 | 1.27 | 0.51 | 1.00 | 1.29 | 0.02 | 0.06 | 0.23 | 0.15 |
| %CV | 2.98 | 2.63 | 4.00 | 2.65 | 3.72 | 10.30 | 8.00 | 6.03 | 4.43 | 1.49 |
Fig. 5.
MRM chromatograms from a human serum sample showing daidzein (27.94 ng/mL), genistein (27.54 ng/mL), equol (24.68 ng/mL), DHD (32.51 ng/mL), O-DMA (19.60 ng/mL) and IS (200 ng/mL).
CONCLUSION
An accurate, reproducible and ultra-fast LC-MS/MS assay for the quantification of phytoestrogens in human plasma has been developed and validated. The assay permits analysis over a range of 1–5000 ng/mL of phytoestrogens using 200 µL of aliquots of human plasma with precise and accurate quantification. Using this method, we have now analyzed over two thousand serum samples, indicating that this assay has adequate sensitivity and specificity to perform high-throughput analysis of a large number of samples obtained from epidemiological studies.
ACKNOWLEDGMENTS
These studies were supported by a grant-in-aid to the Purdue-UAB Botanical Center for Age-related Disease by NCCAM (5P50 AT-00477, C. Weaver, PI) and by the NIA to UCLA (1 R01 AG030448, G. Greendale, PI). Operation of the UAB Targeted Metabolomics and Proteomics Laboratory has been supported in part by a NCI Core Research Grant to the UAB Comprehensive Cancer Center (NCI P30 CA13148-35). The triple quadrupole mass spectrometer was purchased from funds provided by the University of Alabama at Birmingham Health Services Foundation General Endowment Fund.
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