determination of lte4 in human urine by liquid chromatography coupled with ionspray tandem mass...

JOURNAL OF MASS SPECTROMETRY, VOL. 31, 987-993 (1996)

Determination of LTE4 in Human Urine By Liquid Chromatography Coupled With Ionspray Tandem Mass Spectr ometr y

Yunhui Wu, Lily Y.-T. Li and Jack D. Henion* Advanced BioAnalytical Services, Inc., 15 Catherwood Road, Ithaca, New York, New York 14850, USA

George J. Krol Bayer Corporation, 400 Morgan Lane, West Haven, Connecticut 06516. USA

A sensitive and specific high-performance liquid chromatographic/ionspray tandem mass spectrometric (LC/ionspray MS/MS) method was developed and validated to quantitate leukotriene E, (LTE,) in human urine. This method involves solid-phase extraction with Empore membrane disks to isolate LTE, and its internal standard, LTE,-d, , from the urine stabilized with antioxidant and metal ion chelating agent. The reconstituted extracts were analyzed by LC/ionspray MS/MS in the selected reaction monitoring (SRM) mode. The assay has a lower level of quantitation (LOQ) of 50 pg m1-l for LTE, based on 5 ml aliquots of urine. The calibration graphs were linear from 50 pg ml-' to 10 ng ml-' for LTE, extracted from urine. The inter- and intra-assay precision (RSD) and accuracy (DEV) did not exceed 11% and 8% at any level of the calibration standards and quality control (QC) samples, respectively. The recovery of LTE, using the Empore disk solid-phase extraction technology was independent of LTE, concentration in human urine. The overall extraction recovery for LTE, was 72% (RSD = 2.14%).

KEYWORDS: leukotriene E,; quantitation; LC/MS/MS; solid-phase extraction

INTRODUCTION

Leukotrienes (LTs) are biologically important compounds derived from arachidonic acid by the action of the enzyme 5-lipoxygenase. LTE, is a sulfidopeptide leukotriene that is difficult to quantitate because it is relatively unstable under a variety of common experimental conditions. Recently, the determination of LTE, in human and rabbit urine has been reported using solid-phase extraction (SPE)/high-performance liquid chromatography (HPLC)/enzyme immunoassay (EIA) and HPLC/UV detection, respectively.'*' In both methods, EDTA was used to chelate metal ions which might catalyze oxidative processes of sulfidopeptide leu- k~tr ienes .~ 4-Hydroxy-TEMPO (4-hydroxy- 2,2,6,6- tetramethylpiperidinyloxy) was also used in one of the methods' as an antioxidant. The extraction recoveries of LTE, were 58.5% and 76.3% from human and rabbit urine, respectively. A detection limit of 2 ng ml-' for LTE, was reported.' A sufficiently sensitive but a rather complicated gas chromatographic/mass spectrometric (GC/MS) procedure for the determination of LTE, in urine has also been reported., Direct determination of LTE, in urine by GC/MS is virtually impossible since LTE, is thermally labile and non-volatile. There- fore, LTE, was converted into pentafluorobenzyl (PFB) ester trimethylsilyl (TMS) ether derivative of 5- hydroxyeicosanoic acid by catalytic reduction/

* Author to whom correspondence should be addressed.

desulfurization with hydrogen gas followed by treatment with PFB bromide and N,O-bis(trimethylsily1) trifluoroacetamide (BSTFA).,

Mass spectrometry has played an important role in the study of le~kotrienes.~ Its unique capabilities provide detailed information about structure and reliable and specific information for quantitation. Recent developments in mass spectrometry, especially electro- spray ionization (ESI), have made the coupling between liquid chromatography and mass spectrometry SUE- ciently dependable for reliable routine sample analysis.6-" With the very high selectivity and specificity provided by tandem mass spectrometry, LC/MS/ MS has become a powerful technique for pharmaceutical analyses and quantitation.

We have developed a highly sensitive, specific and simple LC/MS/MS method to quantitate LTE, in human urine. This method involves solid-phase extraction with Empore membrane disks to isolate LTE, and its internal standard, LTE,-d3 (Fig. l), from human urine stabilized with 4-hydroxy-TEMPO antioxidant

R=H,LTE4 MW=439 R=D,LTE,d, MW=442

Figure 1. Structures of LTE, and LTE,-d3.

CCC 1076-5174/96/090987-07 0 1996 by John Wiley & Sons, Ltd.

Received 26 March 1996 Accepted 17 May 1996

988 Y. WU ET AL.

and EDTA metal ion chelating agent. Reconstituted extracts were analyzed by LC/MS/MS operated in the negative-ion mode using an ionspray LC/MS interface.

EXPERIMENTAL

Materials and chemicals

LTE, (purity 99.5%) and LTE,-d, (purity 98%) were obtained from BIOMOL Research Laboratories, (Plymouth Meeting, PA, USA). Acetic acid was obtained from J.T. Baker (Danvers, MA, USA), methanol from Baxter Scientific Products (McGaw Park, IL, USA), ammonia solution from EM Science (Gibbstown, NJ, USA) and ethylenediaminetetraacetic acid (EDTA) disodium salt dihydrate and 4-hydroxy-TEMPO from Aldrich Chemical (Milwaukee, WI, USA). Empore CIS extraction disks were obtained from 3M Industrial and Consumer Sector (St Paul, MN, USA). The reagents and materials were obtained from the sources noted or from equivalent sources. The source was not believed to affect assay performance. All chemicals were of HPLC- grade (liquids) or ACS grade (solids).

LC/MS/MS instrumentation

A Hewlett-Packard (Avondale, PA, USA) Model 1090L HPLC pump was used. Chromatography was performed on a Kromasil C, column (50 x 2 mm i.d., 5 pm) (Keystone Scientific, Bellefonte, PA, USA) using isocratic elution with methanol-aqueous ammonium acetate (pH 5.6) (70: 30). The pH 5.6 eluent was prepared by mixing 1000 ml of HPLC-grade water, 0.8 ml of glacial acetic acid and 0.4 ml of concentrated ammonia solution followed by adjusting the pH to 5.6 with glacial acetic acid or concentrated ammonia solution (ACS reagent). The flow rate was maintained at 200 pl min-' for all LC/MS/MS experiments, and the total eMuent was directed to the ionspray interface. A PE- SCIEX (Thornhill, Ontario, Canada) API I11 + tandem triple-quadrupole mass spectrometer equipped with the TurboIonSpray option was used for all analyses. The ionization interface used was ionspray operated in the negative-ion mode. Mass calibration was performed daily by infusion of a lo-, M PPG (polypropylene glycols, average M, 425, 1000 and 2000) calibration solution (PE-SCIEX API 111 + operator's manual) at a flow rate of 10 p1 min-'. Mass-axis calibration was performed daily on the ions at m/z 59.0, 325.3, 520.4 and 906.7. Peak widths were -0.6 u at half-height in the single MS mode. Peak widths of the precursor and product ions were -1.2-1.5 u at half-height in all the LC/MS/MS experiments.

All infusion mass spectra were summed over 10-20 scans. The full-scan single MS and product ion mass spectra of the analyte and the internal standard were obtained by infusion of each solution to the mass spectrometer at a flow rate of 10 pl min-' using a Model 55-2226 syringe pump (Harvard Apparatus, South Natick, MA, USA). Full-scan mass spectra were

acquired by scanning Q1 from m/z 100-500 with a scan step of 0.1 u and a dwell time of 2 ms. The product ion mass spectra (MS/MS) were obtained by choosing the molecular anions as the precursor ions at unit mass resolution and scanning 43 from m/z 100-500 with a scan step of 0.1 u and a dwell time of 2 ms.

The selected reaction monitoring (SRM) mode was used for all quantitative LC/MS/MS analyses. The precursor .+ product ions monitored in the negative-ion mode were m/z 438.3+m/z 333 (LTE,) and m/z 441.3 -+ m/z 336 (LTE,-d,). The detailed mass spectrometer conditions used for the LC/MS/MS experiments were as follows: ionspray voltage, -3500 V; orifice voltage, -65 V; declustering potential, -30 V; colli- sion energy, 18 eV; curtain gas (nitrogen) flow rate, 1.6 1 min- ' ; ionspray nebulizer gas (nitrogen) pressure, 70 psi (1 psi = 6.895 kPa); TurboIonSpray gas (nitrogen) flow rate, 4 1 min-'; TurboIonSpray temperature, 500 "C; interface heater temperature, 60 "C; and colli- sion gas (argon) thickness, 2.5 x lo1, atom cm-2.

Sample preparation

Control urine was collected from healthy male subjects. Typically, urine was collected in a 500 ml polypropylene bottle over a period of 2 h. The collected urine was stored at 4°C during the collection period and for an additional period of 4 h at 4°C. Then, 1 ml of 0.4 M 4-hydroxy-TEMPO solution and 1 ml of 0.2 M EDTA disodium solution were added to the urine. Finally, the urine pH was adjusted to 6.5 with concentrated ammonia solution to give the control urine for the preparation of calibration standards and quality control samples. The control urine was stored at - 30 "C.

Stock solutions of LTE, and LTE,-d, (50 pg m1-I from BIOMOL Research Laboratories) were diluted with methanol-water (70 : 30) to give working solutions of 5 pg ml-' and 100 ng ml-', respectively. The concentrations of all solutions were based on the free acids. All LTE, and LTE,-d, working solutions were stored at - 70 "C. Duplicate calibration standards were prepared fresh for each analysis. The concentrations of the standards for the calibration graphs were 0.05, 0.1, 0.25, 0.5,1,2 and 10 ng m1-I of urine for LTE,.

Quality control (QC) samples were prepared from the QC working solutions of LTE, . The QC working solutions were prepared separately by a different analyst to those used to prepare the calibration standards. The concentrations of the low, medium and high quality control samples were 0.1, 1.5 and 5 ng ml-' of urine for LTE, .

Extraction procedure

For each analysis, duplicate 5 ml aliquots of the calibration standard samples, double blank samples, control blank samples and duplicates (for regular analysis) or six replicates (for precision and accuracy analysis) of QC samples were used. Amounts of 5 ng of LTE,-d, were added to each control blank, standard and QC sample. A double blank sample contained neither the test article nor the internal standard. Volumes of 50 pl of methanol water (70 : 30) were substi-

DETERMINATION OF LTE, IN

100

- 6! 75

I v

3

9 %

25 E

tuted for these analytes. A control blank sample contained only the internal standard at the designated level. The pH of the urine samples was adjusted to 4.4-4.6 using aqueous 10% acetic acid immediately before the extraction.

The solid-phase extraction disk was conditioned with 5 ml of methanol and 5 ml of water. The urine sample was then loaded on to the disk. Vacuum was applied, followed by drying the disk for 1 min. After being washed with 5 ml of aqueous 5% formic acid, the disk was dried for another 1 min. Next, the disk was eluted with 0.3 ml of methanol. After being dried for 15 s, the disk was eluted with another 0.3 ml of methanol. The disk was then dried for a final time of 1 min.

The combined methanol eluates were evaporated to dryness under nitrogen in a TurboVap at 45°C for 30 min. The dry residues were reconstituted in 50 pl of methanol-aqueous ammonium acetate (pH 5.6) buffer (70:30, v/v) and then mixed on a vortex mixer for 1 min. The reconstituted extract was transferred to a microsample filter (0.2 pm) (Scientific Resources, Eaton- town, NJ, USA) and centrifuged at 3000 rpm for 5 min at 20 "C. The filtered extracts were then transferred to autosampler vials for LC/MS/MS analysis; 10 p1 of sample were injected by the autosampler.

354.2

238.2 120.0

1 1 5 1 292.1 320.2 423.3 * 441.3 [M-H]' 160.0

I, -

Data acquisition and analysis

Peak area integration was performed by the MacQuan software (version 1.3, PE-SCIEX) residing on a Macin- tosh Quadra 650. Linear regression was performed by our laboratory information management system (DMLIMS + ; PennComp, Wayne, PA, USA). Data for

URINE BY LC/IONSPRAY MS/MS 989

all samples were transferred electronically to an EXCEL spreadsheet for further statistical calculations. All calculations were based on the SRM peak area ratio: the peak area of LTE, was divided by the peak area of the internal standard, LTE,-d, .

RESULTS AND DISCUSSION

The objective of this work was to develop a simple yet sensitive and specific method for the determination of LTE, in human urine. Molecular anions were chosen as the precursor ions in the MS/MS experiments for both the analyte and its internal standard. From the full-scan product ion mass spectra of LTE, [Fig. 2(A)] and LTE,-d, [Fig. 2(B)], it is evident that both compounds produce abundant product ions. It was found that the best sensitivities and minimum interferences are achieved by monitoring transitions at m/z 438.3 --* m/z 333 for LTE, and m/z 441.3 --* m/z 336 for LTE,-d, in the SRM mode. The fragment ions observed at m/z 333 for LTE, and m/z 336 for LTE,-d, are believed to derived from an initial rearrangement of the cysteine side-chain followed by dehydration5

From our experience, the use of the extraction disks was found to have several advantages over the conventional extraction cartridges. First, disks allow much faster flows than the conventional cartridges. The disks also yield good recovery even when a high flow is used during the sample loading and washing periods. For a sample size of 5 ml or higher, restriction on flow would significantly slow the extraction process. Second, owing

333.2

* I

235.2

5,309.m

351.2

I 289.2

317.2 438.3 [M-HJ-

990 Y. WU ET AL.

to the low mass used for the sorbent, elution solvent volumes as small as 100-200 p1 can be used. Therefore, the time-consuming solvent evaporation and reconstitu- tion steps might become unnecessary. This feature is even more important for compounds that are thermally labile during the solvent evaporation process.

The LC conditions were optimized to separate LTE, and LTE,-d, from the endogenous components within a short analysis time. An example of SRM ion current chromatograms of LTE, and LTE,-d, analytical standards is shown in Fig. 3. These data show the response observed from the injection of 5 pg of each standard.

Assay specificity

LTE, is an endogenous component in normal human urine. The endogenous LTE, level in human urine depends on the individual. The endogenous concentration of LTE, could range from several to several hundred picograms per milliliter in urine. However, our treatment of control urine reduced the concentration of endogenous LTE, below the LOQ of this procedure. A representative SRM LCiMS ion current chromatogram for a double blank human urine extract (i.e. containing neither the analyte nor the internal standard) is shown in Fig. 4. A representative SRM LC/MS ion current chromatogram for an extract of control human urine spiked with LTE,-d, at 1 ng ml-' is shown in Fig. 5.

438.3 1333 51

I a 44 1.31336 58

I 1 LTE4-d3 A

Y

0.00 0.50 1 .00 1 .so 2.M) 1 13 26 38 51

Time (min)/Scan

Figure 3. Selected reaction monitoring LC/MS traces of LTE, and LTE,-d, analytical standards. Injection of 5 pg of each standard was made. The Kromasil C, column (50 mm x 2 mm i.d., 5 pm) was eluted with methanol-aqueous ammonium acetate (pH 5.6) (80: 20) at a flow rate of 200 pl min-'.

438.3/333 412

2

L T' ".

441.3/336 136

1001 I

" '

0.00 I .00 2.M 3.00 400 1 26 51 76 101

Time (min)/Scan

Figure 4. Selected reaction monitoring LC/MS trace of control human urine extract. The Kromasil C, column (50 mm x 2 mm Ld., 5 p n ) was eluted with methanol-aqueous ammonium acetate (pH 5.6) (70: 30) at a flow rate of 200 pl min-'.

Figure 6 shows a representative chromatogram from the extract of control human urine spiked with LTE, and LTE,-d3 at 0.05 and 1 ng ml-' levels, respectively. The LC/MS/MS response for LTE, in the control urine (Figs 4 and 5) is negligible compared with that from the low standard at the 0.05 ng ml-' level shown in Fig. 6.

Linearity

In all validation analyses, the linear calibration graphs ranged from 0.05-10 ng ml-' for LTE, in human urine. Linear calibration graphs were con- structed by weighted (l/y) least-squares regression of concentration versus peak area ratio (analyte/internal standard) of the calibration standards. Table 1 sum- marizes the slopes, intercepts and rz values for four vali-

Table 1. Summary of regression equation and r2 for human urine extracts'

No. A (slope) B (intercept) 12

1 0.792 953 0.005 894 0.9987 2 0.738 029 -0.01 3 807 0.9992 3 0.749 488 0.002 746 0.9991 4 0.808656 0.01 1 795 0.9993

a Calibration graph equation, y =Ax + B ; regression method, linear with weighting factor of l/y.

Analysis

DETERMINATION OF LTE, IN URINE BY LC/IONSPRAY MS/MS 99 1

438.31333 459

14 I 3 I336 6919

0.W 1.00 2.W 3.00 4.00 I 26 51 16 101

Time (min)/Scan

Figure 5. Selected reaction monitoring LC/MS trace of an extract of control human urine spiked with LTE,-d, at 1 ng ml-' level. LC/MS/MS conditions as in Fig. 4.

dation analyses from human urine extracts. A representative calibration graph for LTE, extracted from human urine is shown in Fig. 7.

Assay precision and accuracy

The intra- and inter-assay precision and accuracy rep- resent the precision and accuracy observed within an analysis and between different analyses, respectively. The intra-assay precision and accuracy for the method were assessed from the results of both calibration standards and QC samples. Six replicates of each cali-

4-38 3/333 446

J. L. 0.00 1W 2.00 3.m 4.00

1 26 51 76 101 Time (min)/Scan

Figure 6. Selected reaction monitoring LC/MS trace of an extract of control human urine spiked with LTE, at 50 pg ml-' and LTE,- d, at 1 ng ml-'. LC/MS/MS conditions as in Fig. 4.

bration standard level and three replicates of each QC level were used in the intra-assay analysis. The intra- assay precision (RSD) and accuracy (DEV) did not exceed 11% and 4%, respectively, at any level of the calibration standards (Table 2). The intra-assay precision and accuracy did not exceed 4% and 8%, respectively, for any level of QC samples (Table 2). The inter-assay precision and accuracy of the method were assessed from the results obtained with QC samples during nine assay runs. The inter-assay precision and accuracy did not exceed 8% and €240, respectively, at any level of QC samples (Table 3). Table 3 shows the results for each individual QC sample in nine analyses.

Table 2. Summary of intra-assay precision and accuracy for LTE, calibration standards and QC samples

Standard (ng rnl-') CIC (ng rnl-')

Theoretical concentrations 0.050 0.100 0.250 0.500 1.000 2.000 10.000 0.1000 1.500 5.000

Determined 0.054 0.100 0.240 0.484 1.003 2.093 9.828 0.108 1.656 4.968 concentrations 0.049 0.105 0.237 0.527 1.002 2.120 10.045 0.101 1.575 5.082

0.045 0.099 0.237 0.528 1.003 2.034 9.869 0.104 1.596 5.114 0,045 0.123 0.243 0.470 1.063 2.077 9.678 0.048 0.100 0.272 0.482 1.011 2.004 10.133 0.048 0.092 0.241 0.504 1.005 2.044 10.067

Mean 0.048 0.103 0.245 0.499 1,015 2.062 9.937 0.104 1.609 5.055 RSD (Yo) 6.88 10.25 5.48 4.91 2.36 2.06 1.75 3.37 2.61 1.52

n 6 6 6 6 6 6 6 3 3 3 DEV (%) -3.67 3.17 -2.00 -0.17 1.45 3.10 -0.63 4.33 7.27 1.09

992 Y. WU ET AL.

Area(Ra1iol

Figure7. A

LTE4 438.3->333.0

Internal Standard: LTE4d3

Weighted (14) Intercept = 0.002746 Slope = 0.749488 Correlation Coefl. = 0.9991

Area(Aatio)

I 7.00'

6.00'

5.00'

4.00'

#

Cone. (ngimL)

representative calibration graph for LTE, extracted from human urine from 0.05 to 10 ng ml-'

~ ~ ~ ~ ~ _ _ ~~

Table 3. Summary of inter-assay precision and accuracy for LTE, QC samples

Analysis Concentration found (ng ml-')

No.

1 0.1 01 1.667 5.1 39 0.1 08 1.599 6.046 0.092 1.699 5.908

2 0.1 12 1.571 5.1 92 0.1 11 1.603 5.1 53 0.1 08 1.631 5.261

3 0.095 1.545 5.1 65 0.1 06 1.560 5.054 0.1 04 1.558 5.059

4 0.1 06 1.750 5.202 0.1 21 1.690 5.205

5 0.1 18 1.661 5.164 0.1 12 1.672 5.108

6 0.1 11 1.591 4.495 - 1.662 4.956

7 0.098 1.527 4.666 0.088 1.390 4.725

8 0.1 09 1.741 4.578 0.1 05 1.451 5.01 6

9 0.1 08 1.656 4.968 0.1 01 1.575 5.082 0.1 04 1.596 5.1 14

Mean 0.1 06 1.609 5.103 SD 0.008 0.087 0.354 RSD (%) 7.55 5.42 6.94 DEV (%) 5.62 7.26 2.05 n 21 22 22

a Data excluded from statistical calculation owing to low LC/MS/ MS response caused by an injector problem.

QC 1 (0.1 ng ml-') QC 2 (1.5 ng ml-'1 QC 3 (5 ng ml-')

The inter- and intra-assay precision and accuracy obtained satisfied our acceptance criteria which are within 15% for both at each level of the calibration standard and QC sample.

Level of quantitation and recovery

The lower level of quantitation (LLQ) was defined as the lowest concentration on the calibration graph for which an acceptable accuracy of 100 f 20% [(mean observed concentration/theoretical concentration) x 1001 and precision of 20% (RSD) were obtained. The current assay has a limit of quantitation of 0.05 ng ml-I in human urine for LTE, based on 5 ml aliquots of urine. The RSD and DEV at the LOQ were within 7% and 14%, respectively. Although the practical LOQ depends on individual requirements for analyses of bio- logical samples from specific sources, we expected that most of our clinical samples would have LTE, concentrations higher than 50 pg m1-l. SRM LC/MS traces for a representative clinical sample are shown in Fig. 8. The LTE, concentration determined with this sample was 178 pg ml-'.

The extraction recovery of LTE, at three QC levels was determined by comparing the peak area ratio of LTE, to the internal standard LTE,-d3 in samples which were spiked with the analyte prior to extraction (i.e. regular QC samples) with samples to which the analyte was added post-extraction. The internal standard was added to both sets of samples post-extraction. The recovery of LTE, during solid-phase extraction was independent of the LTE, concentration in human

DETERMINATION OF LTE, IN URINE BY LC/IONSPRAY MS/MS 993

4R9 pathway is via reaction of the allylic hydrogen of the triene with free radicals such as hydroperoxide. Both EDTA and 4-hydroxy-TEMPO have been used as metal chelating reagent and antioxidants for the stabili- zation of LTE, in urine. Also reported was the possible isomerization of the double bond at C-11 in highly acidic solutions.'

The stability of LTE, in human urine and in reconsti- tution buffer was also investigated. LTE, was found to be stable in human urine in the presence of 4-hydroxy- TEMPO and EDTA at -70°C for at least 33 days, at ambient temperature up to 24 h and in the reconstitu- tion buffer at 5 "C for 24 h. LTE, was also shown to be stable after three freeze (- 70 "C)-thaw cycles in human urine and to be stable for at least 2 days as dry extracts at - 70 "C.

LTE4 438.3/333

1

441.31336 1500

1MT LTEq-dg

75'

CONCLUSIONS

The present procedure for the quantitation of LTE, in urine has unique aspects. First, the assay procedure is relatively simple. No time-consuming derivatization

oc 0.00 I .oo 2.00 3.00 brane disks significantly reduces the time required for

solid-phase extraction. Finally, excellent sensitivity and selectivity can be achieved using the combination of LC

50

25

steps are required. Second, the use of Empore mem- 1 26 51 76

Time (rnin)/Scnn

Figure 8. Selected reaction monitoring LC/MS trace of an unknown human urine sample spiked with LTE,-d, at 1 ng ml-'. LC/MS/MS conditions as in Fig. 4.

and MSiMs techniques. This-new LC/Ms/MS assay procedure is sensitive, accurate, selective and reproducible. Its high sensitivity allows the reliable and reproducible quantitation of LTE, between 0.05 and 10 ng ml-' in human urine. This fast LC/MS/MS method also allows the analysis of 15 samples per hour. urine. The overall extraction recovery for LTE, was

72% (RSD = 2.14%).

Stability Acknowledgements

We thank Dr Robert C. Murphy of the National Jewish Center for Immunology and Respiratory Medicine for his helpful suggestions during the method development phase of this work.

It has been reported that metal ions might catalyze the oxidation process of LTE,.3 Another possible oxidation

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determination of lte4 in human urine by liquid chromatography coupled with ionspray tandem mass...

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