Clinical Chemistry 53: 251-258, 2007.
First published January 2, 2007; 10.1373/clinchem.2006.074989
(Clinical Chemistry. 2007;53:251-258.)
© 2007 American Association for Clinical Chemistry, Inc.
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Lipids, Lipoproteins, and Cardiovascular Risk Factors |
Isoprostane Measurement in Plasma and Urine by Liquid Chromatography–Mass Spectrometry with One-Step Sample Preparation
Debajit Sircar and
Papasani V. Subbaiaha
1 Departments of Medicine and Biochemistry and Molecular Genetics, University of Illinois at Chicago, Chicago, IL.
aAddress correspondence to this author at: Department of Medicine, 1819 West Polk St., M/C 797, Chicago, IL 60612. Fax 312-413-0435; e-mail: psubbaia{at}uic.edu.
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Abstract
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Background: Isoprostane F2
(iPF2-III) concentration in plasma and urine is widely accepted as a measure of oxidative stress. Gas chromatography–mass spectrometry (GC/MS) methods for measuring iPF2-III involve several steps of sample preparation and are labor-intensive, and ELISA methods, although easier to use, are less reliable. Therefore we developed a simple and sensitive method involving 1-step sample cleanup and HPLC/MS quantification.
Methods: Samples of plasma or urine were enriched with a deuterated (iPF2-III-D4) standard, treated with KOH to liberate the bound isoprostanes, then loaded onto an immunoaffinity column, and the bound isoprostane was eluted with 95% ethanol. The concentrated sample was injected onto a C-18 HPLC column, and the isoprostane was eluted with a gradient of acetonitrile in water and analyzed by electrospray negative ionization, selectively monitoring the ions 353.2 (iPF2-III) and 357.2 (iPF2-III-D4). The amount of isoprostane in the sample was calculated from the ratio of the intensities of the 2 ions.
Results: The described method has a detection limit of 0.5 ng/L, with a linear dynamic range of 1–5000 ng/L. The intra- and interassay imprecisions were 4.68% and 3.88%, respectively. The values obtained correlated strongly with the GC/MS procedure (r = 0.80), but the absolute values were 
4– to 5-fold lower, because the present method measures specifically 1 isomer of isoprostane, whereas the GC/MS method measures 4 isomers together.
Conclusions: Because of its simplicity and lower limit of quantification, the present method provides a useful noninvasive tool for determining oxidative stress in patients.
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Introduction
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Isoprostanes, the stable end products of peroxidation of arachidonic acid, are widely accepted as reliable indicators of oxidative stress in vivo (1)(2). Although theoretically 64 possible isomers can be generated by the oxidation of arachidonic acid, 8-isoprostaglandin F2 (iPF2-III;1
also known as 15 F2t-isoprostane) is commonly used as a marker of oxidative stress (2). The most commonly used methods for measuring isoprostaglandin F2-III (iPF2-III) are gas chromatography–mass spectrometry (GC/MS) (2)(3)(4)(5) and ELISA (6)(7)(8). Although immunoassay methods are easier to use and require less expensive instrumentation, controversy exists regarding their specificity and correlation with the widely accepted gold standard GC/MS methods (2)(8). On the other hand, the GC/MS methods involve multiple steps, including extensive sample preparation, derivatization, and cleanup, that are not only labor-intensive but also lead to contamination, artifact generation, and poor recoveries. Liquid chromatography (LC)/MS methods have been developed that do not require a derivatization step and therefore are less prone to artifacts and loss of material (2)(9)(10)(11).
Ohashi and Yoshikawa (9) reported a method for the determination of iPF2 in plasma and urine, which involves solid phase extraction (SPE) followed by LC/electrospray ionization (ESI)/MS in selective ion monitoring (SIM) mode. Although the limit of detection (LOD) was reported to be 2 ng/L plasma, the chromatogram contained several peaks of the ion of interest, of which only a single minor peak corresponded to the standard iPF2. Furthermore, this study reported only the estimation of free (unbound) isoprostane concentrations, a method that would not be suitable for an accurate assessment of the oxidative stress in vivo because most plasma isoprostanes are bound to lipids (12)(13)(14). Several groups have also reported the estimation of isoprostanes with LC/tandem MS (LC/MS/MS) (2)(10)(11). The most detailed of these methods, reported by Liang et al. (10), employs SPE for sample preparation and LC/MS/MS for the estimation of urinary isoprostanes. The report of this method, however, did not address total isoprostane measurement and did not determine the suitability of the method for plasma. The LOD was rather high (9 pg), and the chromatogram contained 9 peaks, of which only a minor peak corresponded to the standard iPF2. Similarly, the method of Coolen et al. (11), which also used SPE for cleanup, showed several peaks of the desired ion pair (m/z 353/193) in plasma samples. Because of our interest in measuring total isoprostane concentrations in the plasma and urine, we developed a method that uses the less expensive single quadrupole equipment and combines the simple sample preparation of immunoassays with the sensitivity and selectivity of the LC/MS method. Our method involves liberation of the bound isoprostanes with mild alkaline hydrolysis, followed by their isolation with single-step immunoaffinity chromatography (IAC). The resulting preparation gives a single molecular ion peak (353 m/z) in SIM mode and is detectable at <0.5 pg.
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Materials and Methods
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materials
Isoprostane immunoaffinity columns (4 mL volume, containing 0.5 mL resin) were purchased from Cayman Chemical. The binding capacity of each column, according to the manufacturer, was 5 ng isoprostane. Standard iPF2-III (9,11,15S-trihydroxy(8ß)prosta-5Z,13E-dien-1-oic acid), deuterated iPF2III (3,3,4,4-D4 analog), and the 3H-radiolabled iPF2-III (9-3H) were all purchased from Cayman Chemical and were used without further purification. All solvents were of HPLC grade and were purchased from Fisher Scientific Co. Plasma samples were obtained from the local blood bank (Life Source Inc., Glenview, IL). The blood was collected with the anticoagulant citrate/phosphate/dextrose, and after separation of the plasma by centrifugation, it was shipped to the laboratory at 4 °C within 24 h. Glutathione (1 mg/mL) and BHT (0.05%) were added to the plasma, and 1-mL aliquots of plasma were stored at –80 °C until use. For the purpose of method evaluation, plasma samples (from anonymous donors) were also obtained from Dr. Laurie Quinn (University of Illinois) and Dr. Michael Davidson (Rush Medical Center). These samples were originally collected in EDTA as anticoagulant, contained glutathione (1 mg/mL) and butylated hydroxytoluene (0.05%), and were stored at –80 °C. Urine samples (random) from healthy donors (4 males, 7 females, ages 21–55 years) were also supplied by Dr. Laurie Quinn (University of Illinois), and stored at –80 °C until use. Anonymized urine samples containing known amounts of isoprostanes (as measured by GC/MS) were kindly provided by Drs. Ginger Milne and Jason Morrow of Vanderbilt University, for method validation.
isoprostane extraction from plasma
The final procedure adopted for the isolation and analysis of isoprostanes from plasma is shown in Scheme 1 (see Scheme 1 in the Data Supplement that accompanies the online version of this article at http://www.clinchem.org/content/vol53/issue2 ). To 0.3 mL of freshly thawed plasma, 200 pg of deuterated internal standard (iPF2-III-D4) and 0.3 mL of 15% KOH were added, and the sample was incubated for 60 min at 40 °C. The alkali was neutralized (to pH 7.2–7.4) by the addition of 1 mL of 1 mol/L KH2PO4 and was loaded onto the isoprostane affinity column, which had been prepared according to the manufacturer’s instructions. The column was washed with 5 mL of 0.1 mol/L phosphate buffer and 5 mL of ultrapure water, and then the isoprostanes were eluted with 2 mL of 95% ethanol. The eluate was evaporated to dryness under vacuum in a SpeedVac (Savant Instruments), and the sample was redissolved in 60 µL of 20% acetonitrile in water, containing 1 mg/mL acetic acid. We injected 20 µL of the sample into the HPLC system and analyzed it as described below. The affinity column was regenerated by washing with 5 mL of ultrapure water and 5 mL of 0.1 mol/L phosphate buffer containing 0.05% NaN3.
isoprostane extraction from urine
The urine sample (0.3 mL) was first diluted with 2 mL of 0.1 mol/L KH2PO4 buffer, pH 7.4, enriched with 200 pg of deuterated iPF2, and directly loaded onto the affinity column without the KOH treatment. The elution of the isoprostane was exactly as described above for plasma samples.
lc/ms analysis
The equipment used for the LC/MS analysis was the Surveyor HPLC System from Thermo Finnigan, interfaced with a Surveyor MSQ single quadrupole mass spectrometer. The HPLC system consisted of a quaternary pump, an autosampler with temperature-controlled sample compartment, a degasser, and a C-18 column (Alltech Altima HP C18, 100 mm x 2.1 mm, 3 µm particle size), and a guard column (10 mmx 2.1 mm). The mass spectrometer was equipped with a single quadrupole mass analyzer, an ESI probe, a turbomolecular pump, and a cone-wash system. The HPLC system and the mass spectrometer were controlled by XCaliber 1.4 software for Windows (Thermo Finnigan).
The sample was chromatographed with a linear gradient of acetonitrile in water (20% to 45% in 25 min) at a flow rate of 200 µL/min. The column was then equilibrated back to the initial condition (20% acetonitrile) in 20 min. The total time from injection to injection was 45 min. The concentration of acetic acid was maintained at 50 g/L throughout the run, and the column temperature was maintained at 30 °C. The sample tray temperature was kept at 4 °C. A flow diverter was used to divert the column eluate to waste from 0 to 15 min and from 25 to 45 min of the run. A continuous cone wash was applied with ultrapure water containing 50 g/L acetic acid, at a flow rate of 200 µL/min.
The mass spectrometer conditions were as follows: negative ESI mode, drying gas flow (N2) at 650 L/h, needle voltage at 2.3 KeV, probe temperature at 400 °C, cone voltage at 60 eV, and detector voltage at 1953 V. The sample was analyzed in SIM mode for the molecular ions of iPF2 (m/z 353.2) and the deuterated internal standard (m/z 357.2). The concentration of isoprostane in the sample was calculated from the area ratio of the peaks m/z 353.2 and m/z 357.2.
creatinine estimation
Creatinine concentration in urine samples was measured enzymatically with a reagent set from Cayman Chemical Co.
ex vivo oxidation of plasma
For the determination of isoprostane generated during ex vivo oxidation of plasma, 0.3 mL of normal plasma was diluted with 1.8 mL of phosphate buffer, pH 7.4, and incubated with 50 µmol/L CuSO4 at 37 °C for various periods of time. The oxidation was stopped by the addition of 0.6 mmol/L EDTA, and the isoprostane concentration was measured by the present method after addition of the deuterated standard. Duplicate samples were oxidized under identical conditions, their lipids were extracted by Bligh and Dyer procedure (15), and the conjugated diene concentration was determined by absorbance at 234 nm in a spectrophotometer.
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Results and Discussion
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isoprostane extraction from human plasma
Most of the methods for the determination of isoprostanes by GC/MS and LC/MS use SPE cartridges to isolate iPF2-III from the plasma or urine samples. An examination of the published procedures for LC/MS, however, shows multiple peaks in the chromatograms even when single ions or ion pairs are monitored. This finding is attributable to the presence of several closely related eicosanoids and prostanoids in the plasma and urine that behave similarly on solid phase columns. Moreover, alkaline hydrolysis of plasma to liberate the bound isoprostanes could generate further artifacts. Therefore, we sought to use immunoaffinity columns, which are commercially available and are known to be highly specific for the iPF2-III.
We followed the protocol suggested by the manufacturer of the columns, with a few modifications. First, because most of the plasma isoprostanes are present in the esterified form, we used alkaline hydrolysis to liberate the isoprostanes. Second, the ethanol extraction step was omitted after the alkaline hydrolysis. Instead, the plasma sample was neutralized directly with KH2PO4 and loaded onto the affinity column after dilution. We also used increased column wash volume, which resulted in higher resolution peaks in LC/MS. The neutralization of the plasma to the correct pH (7.2–7.4) after alkaline hydrolysis was found to be critical in the efficient binding and recovery of the isoprostanes.
extraction efficiency and specificity of the immunoaffinity columns
The manufacturer recommends the use of the affinity columns up to 4 times for the extraction of 200 µL of plasma and only once for 1 mL urine. These recommendations, however, were for the assay of free isoprostanes in plasma. Because our procedure involved alkaline treatment of plasma, the efficiency of the antibody could be further affected. To determine the effectiveness of the columns for repeated use, we enriched the plasma or urine samples (0.3 mL) with 3H-labeled iPF2-III (21 000 dpm), and determined the recovery of the label after repeated use of the same column. In addition, we estimated the endogenous iPF2 concentration with the help of the deuterated internal standard. As shown in Fig. 1
, the recovery of labeled isoprostane from plasma decreases substantially after the first use, but the losses were less significant in subsequent extractions. The calculated recoveries, however, were excellent for at least 3 extractions, because of the correction for the losses by the use of deuterated internal standard. After 3 extractions, however, the variability of recovery was much higher with different columns. In the case of urine samples, for which the alkaline hydrolysis was omitted, the columns could be used at least 5 times without significant deterioration of absolute recovery (as measured by radioactivity) or calculated recovery (as measured by LC/MS with internal standard). Therefore, we recommend the use of the columns up to 5 times with 0.3 mL or less of urine sample and up to 3 times with 0.3 mL or less of plasma sample. According to the manufacturer, the antibody cross-reacts weakly with 8-isoprostaglandin F3 (7.6%) and with prostaglandin F1 (2.85%). Because 8-isoprostaglandin F3 (FW 352.5) and prostaglandin F1 (FW 356.5) are not isobaric with iPF2-III (FW 354.5), these compounds would not interfere in our assay even if they were present in the sample after the affinity column step. Furthermore, the gradient conditions used for HPLC should separate most of the interfering eicosanoids from iPF2-III (9)(10). Studies by Tsikas et al. (14) also showed that 15 (R) isomer of iPF2 does not bind to the affinity columns used in the present study and therefore can be excluded as a possible contributor to the measured values.
recovery, linearity, limits of detection and quantification, and imprecision
Typical SIM chromatograms for iPF2-III from plasma and urine samples enriched with deuterated internal standard are shown in Fig. 2
. A single peak of the expected mass was obtained in both plasma and urine samples, corresponding to the internal standard. The immunoaffinity purification of the sample therefore yielded a much cleaner preparation than the SPE procedures, which result in multiple peaks even when analyzed by tandem MS (10)(11). Although occasional plasma samples showed the presence of other m/z 353 peaks, they were well separated from the iPF2-III peak under the HPLC conditions used.
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Figure 2. SIM chromatograms obtained from analysis of plasma and urine samples enriched with deuterated iPF2-III.
Chromatography conditions were as described in the text. Samples were monitored for m/z 353.2 and m/z 357.2
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Recovery of the method for analysis of biological samples was determined by analyzing increasing amounts of standard iPF2-III in the presence and absence of biological sample (plasma or urine). We used 2 plasma samples, one with a low isoprostane concentration and the other with a high concentration. The samples were enriched with 660 pg of deuterated standard and 0–2000 ng/L of iPF2-III, and the isoprostanes were quantified. Corresponding concentrations of iPF2-III standards were also directly injected into the LC/MS without affinity column purification. The urine samples were similarly enriched with 660 pg of deuterated internal standard and 0–3300 ng/L of iPF2-III, and analyzed as described in Materials and Methods. As shown in Fig. 3
, the slopes of detector responses were similar in the presence and absence of plasma or urine matrix. The calculated recoveries of the enriched sample at various concentrations were 78%–102% for the plasma matrix and 75%–99% for the urine matrix. The low recovery occurred at the lowest concentration of enriched iPF2-III. The extrapolated values were 40.4 ng/L for the low isoprostane plasma, 368.6 ng/L for the high isoprostane plasma, and 361.3 ng/L for urine. The corresponding values measured directly in the unenriched samples were 37.6, 356.8, and 380 ng/L, respectively. The lower limit of quantification (LOQ) (defined as the lowest concentration added that can be measured with <20% error) was 30 ng/L plasma or urine.
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Figure 3. Analytical recovery of the method.
(A), recovery of standard iPF2-III added to plasma. Plasma 1 (low isoprostane concentration, 0.3 mL) was enriched with 200 pg of deuterated iPF2-III and 0, 10, 20, 50, 100, 300, or 600 pg of unlabeled iPF2-III, and plasma 2 (high isoprostane concentration, 0.5 mL) was enriched with 200 pg of deuterated standard and 0, 20, 50, 100, 300, or 600 pg of PF2-III. Corresponding amounts of isoprostane standards were also directly injected and analyzed by LC/MS to calculate the recoveries. The isoprostane values are shown per milliliter of plasma. The extrapolated value for unenriched plasma 1 was 40.4 ng/L, and for plasma 2, it was 368.6 ng/L. The actual estimated values were 37.6 ng/L and 356.8 ng/L, respectively. (B), recovery of isoprostane added to urine sample. The urine sample (0.3 mL) was enriched with 200 pg of deuterated iPF2-III, and 0, 20, 50, 100, 600, or 1000 pg of unlabeled iPF2-III and analyzed by LC/MS as described in the text. Standard isoprostane corresponding to the above concentrations was also injected directly into LC/MS to calculate recoveries. The extrapolated value for unenriched urine was 361.3 ng/L, and the actual measured value was 380 ng/L.
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The linearity of the assay for unenriched plasma and urine samples is shown in Fig. 1 in the online Data Supplement. The plasma sample used here was not stored in the presence of antioxidants, and therefore the values were higher than the average values for the normal human plasma. Nevertheless, at least 0.5 mL of plasma or urine could be analyzed under linear conditions by the described method. The minimum amount of plasma sample required is 100 µL, which is lower than that required for the GC/MS analyses, most of which require 1–3 mL plasma (4)(16)(17)(18). We recommend the use of 0.2–0.3 mL of plasma with the present method for better results. Larger volumes of plasma can be used if the isoprostane values are low, but the efficiency of the affinity column is correspondingly decreased after repeated use.
The LOD was 0.5 pg per injection at a signal-to-noise ratio of 3. The LOQ for the standard isoprostane was 1.0 pg per injection at a signal-to-noise ratio of 10. The precision of LOQ was 8.25% (CV). The linear dynamic range was 1–5000 pg per injection (see Fig. 2 in the online Data Supplement).
The imprecision of the method was determined by estimating the iPF2-III values in 2 plasma samples in triplicate, each on 3 different days, and 1 urine sample in triplicate on 4 different days. The intraday imprecision (CV%) was 4.68 for plasma (n = 18; mean value, 68.6 ng/L) and 3.83 for urine (n = 12; mean value, 365 ng/L). The interday imprecision (CV) was 3.9 for plasma (n = 6) and 2.98 for urine (n = 4).
comparison of the present method with the gc/ms method
To determine the correlation of the LC/MS method used here with the more commonly used GC/MS procedure, we obtained 10 urine samples whose isoprostane concentration was determined by GC/MS by the reference laboratory at Vanderbilt University, through the courtesy of Drs. Ginger Milne and Jason Morrow. The values obtained with LC/MS assay were 4- to 5-fold lower than those obtained by the GC/MS procedure (Fig. 4
). The difference in absolute values is most likely due to the estimation of multiple isomers of iPF2 by the GC/MS procedure, as shown by several studies (14)(16). On the other hand, the present method determines specifically 15 (S) iPF2. Studies by Tsikas et al. (14) showed that the purification of isoprostane by IAC results in specific extraction of 15 (S) iPF2 isomer, and that the amount of isoprostane (determined by GC/MS/MS) was less than half of that obtained after SPE/thin-layer chromatography purification steps. In contrast, Liang et al. (10) reported that the amount of urinary iPF2-III estimated by LC/MS/MS after SPE purification and IAC purification was similar. Although we cannot completely exclude the presence of other unknown isomers, it is clear that IAC results in the estimation of predominantly 1 isomer of isoprostane. Furthermore, even after SPE extraction, the values obtained by LC/MS procedures are much lower than those obtained by GC/MS because of the ability of LC columns to separate the underivatized isomers (10). We have also analyzed the IAC eluates of 5 urine samples by MS/MS as described by Liang et al. (10) (see Fig. 3 in the online Data Supplement). There was good correlation between the 2 determinations, although the LC/MS/MS values were slightly higher than the LC/MS values. Therefore, the more expensive LC/MS/MS is not necessary for measurement, if the samples are first purified by IAC.
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Figure 4. Correlation with GC/MS.
Urine samples containing known amounts of isoprostane, as determined by the GC/MS assay (1) in a reference laboratory (Vanderbilt University), were analyzed by the present method. The left panel shows the correlation of the absolute values obtained by the 2 methods. The right panel shows the difference (Bland–Altman plot) between the averages of the 2 measurements (GC/MS + LC/MS /2) and the percent differences [(GC/MS-LC/MS)/Average · 100]. The 95% confidence limits (± 2 SD) are also shown.
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ex vivo oxidation of plasma
To further demonstrate the applicability of the method to biological samples, we oxidized a sample of human plasma in the presence of 50 µmol/L CuSO4 for various periods of time and used the present method to measure the amount of isoprostane generated. In duplicate samples of similarly treated plasma, the formation of conjugated dienes was estimated by measuring the absorbance at 234 nm in the total lipid extract, as described in Materials and Methods. As shown in Fig. 4 in the online Data Supplement, the 2 measures of oxidation showed similar patterns during the course of oxidation, showing that the isoprostane quantification by the present method correlates with the extent of lipid peroxidation in plasma.
comparison of plasma and urine isoprostane values with literature values
The values obtained with different methods of analysis for plasma and urine from apparently healthy donors are shown in Table 1
. It is evident that even with GC/MS, the reported values differ from laboratory to laboratory, indicating the variability in the number of isomers measured and the purification of the samples. The values reported by the present method fall within the range of values reported for GC/MS but are closer to the values reported with LC/MS/MS (11). In addition to the differences in patient populations, the extraction procedures used and the efficiency of column separation of isomers and contaminants could contribute to these variabilities among laboratories.
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Table 1. Comparison with literature values for plasma and urine isoprostane concentrations (in healthy controls, except where indicated).
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Conclusions
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This method combines the simplicity and specificity of IAC for sample preparation with the sensitivity and selectivity of LC/MS for quantification. Compared with the more widely used GC/MS procedures, the present method involves fewer steps, and because of the omission of derivatization and clean-up steps, it is less prone to sample losses and artifact generation. The LLOQ is better than most of the GC/MS methods, and the method requires only 0.3 mL of urine or plasma. Because of the purification of the sample by IAC, the more expensive triple-quadrupole MS equipment is not required. Because the isoprostanes have emerged as one of the most important noninvasive markers of oxidative stress associated with the development of several diseases, including heart disease, cancer, and Alzheimer, the method fills an important need for the clinical laboratory.
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Acknowledgments
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This study was supported by NIH Grant HL 68585. We thank Drs. Laurie Quinn and Michael Davidson for providing plasma and urine samples and Drs. Ginger Milne and Jason Morrow of Vanderbilt University for providing the urine samples with known concentration of isoprostanes measured by GC/MS. We also thank Drs. Evgeny Berdyshev and V. Natarajan (University of Chicago) for the analysis of samples by LC/MS/MS.
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Footnotes
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1 Nonstandard abbreviations: iPF2-III, isoprostane F2; iPF, isoprostaglandin F; GC, gas chromatography; MS, mass spectrometry; LC; liquid chromatography; LOD, limit of detection; SPE, solid phase extraction; ESI, electrospray ionization; SIM, selective ion monitoring; IAC, immunoaffinity chromatography; LOQ, limit of quantification. 
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Abstract
Introduction
