Journal of Analytical Toxicology, Vol. 31, April 2007

Measurement of Aflatoxin and Aflatoxin Metabolites in Urine by Liquid Chromatography-Tandem Mass Spectrometry"*

Robert A. Everley 1,2, Frederic t. CineP, Dongliang Zhang 1, Peter F. ScholP, John D. Groopman 3, and Timothy R. Croley 1,2,* 1Commonwealth of Virginia, Division of Consolidated Laboratories, 600 N. 5th Street, Richmond, Virginia 23219; 2Virginia Commonwealth University, Department of Chemistry, 1001 W. Main Street, P.O. Box 842006, Richmond, Virginia 23284-2006; 3Department of Environmental Health Sciences, Bloomberg School of Public Health, Johns Hopkins University, Baltimore, Maryland 21205-2103

I Abstract

Automated immunoaffinity solid-phase extraction followed by liquid chromatography-tandem mass spectrometry and chemical analogue internal standardization is employed to detect and quantify the aflatoxins AFB1, AFB2, AFG1, AFG2, and the metabolites AFM1 and AFP 1 in urine. The dynamic range of the method is nearly three orders of magnitude with limits of detection in the low femtogram on column range. The method was validated over a 12-day period by eight analysts. This method is suitable for agricultural, forensic, and public health laboratories during an accidental outbreak or a chemical terrorism event where a rapid and accurate diagnosis of aflatoxicosis is needed.

Introduction

Because of the ever increasing threat of terrorist attacks around the globe and, more specifically, the threat of a chem- ical terrorism attack, analytical chemistry laboratories that would aid in forensic investigations and public health domains must be prepared to provide quality laboratory results quickly and efficiently. In such an event, the number of victims could be large and the type of warfare agent may not be immediately obvious. To this end, analytical methods that can provide rapid and sensitive confirmation and quantitation of the agent are vital in determining which agent is used, each individual's de- gree of exposure, and the extent of the population that is ex- posed (1).

* Results from this research were presented at the 54th ASMS Conference on Mass Spectrometry. t Author to whom correspondence should be addressed: Timothy R. Croley, Commonwealth of Virginia, Division of Consolidated Laboratories, 600 N. 5th St. Richmond, VA 23219. E-mail: tim.croley@dgs.virginia.gov.

Of the many toxins that could be used in an attack, those previously weaponized are of particular interest. One example is aflatoxins, which were weaponized by the Iraq government during the first Gulf War. According to a United Nations Spe- cial Commission (UNSCOM) report, the Salman Pak weapons facility in Iraq produced 2200 L of aflatoxins loaded in 122-mm rockets, 400-1b bombs, and SCUD missiles (2).

Aflatoxins are secondary metabolites of the fungi Aspergillus flavus, from which their name (A. fla.) is derived. The pre- dominant aflatoxins AFB1, AFB2, AFG1, and AFG2 are desig- nated B and G because of their blue and green fluorescent color observed under UV illumination. The most studied and most hepatotoxic aflatoxin is AFB1 (LDs0 1.16 mg/kg in rat) (3), for which the World Health Organization (WHO) suggests there is no safe dose (4). Moreover, these compounds are known to be mutagenic and teratogenic. Clinical symptoms of aflatoxin exposure include abdominal pain, rash, and gas- trointestinal bleeding (5,6). The commonality of these symp- toms with those seen in other illnesses prevent them from providing unambiguous identification of their cause, which further emphasizes the need for an analytical method that provides more definitive information and enables a conclusive diagnosis.

The work presented here builds upon previous investigations of aflatoxins in urine (7,8) in an effort to improve both the sen- sitivity and dynamic range of those methods. To improve upon the speed of previous methods, an automated immunoaffinity solid-phase extraction (SPE) method has been developed in conjunction with liquid chromatography-tandem mass spec- trometry (LC-MS-MS) analysis to take advantage of the sen- sitivity, specificity, and ease of quantitation the technique provides. Figure 1 shows the structures for the four parent aria- toxins of interest and the metabolites of AFB1 that were chosen for this study.

150 Reproduction (photocopying) of editorial content of this journal is prohibited without publisher's permission.

Journal of Analytical Toxicology, Vol. 31, April 2007

Experimental

Chemicals and materials High-performance liquid chromatography (HPLC) grade

methanol, acetonitrile, and formic acid were purchased from Fisher Scientific (Fairlawn, N J). Deionized water was purified in house to yield organic-free 18.3 M I x cm water using an E- pure purification system (Barnstead International Dubuque, IA). Aflatoxin reference standards (AFB1, AFB2, AFG1, AFG2, AFM1, and AFP 1) were purchased from Sigma-Aldrich Chem- ical Company (St. Louis, MO).

Standard preparation and characterization Aflatoxins and the internal standard AFB2 were dissolved in

acetonitrile and diluted in 85:15 MeOH/H20 (v/v) to a final con- centration of 1 ng/pL. Eight calibration standards, low and high quality controls (QCs), and a urine blank containing in- ternal standard were stored in 50-mL polypropylene conical tubes at 4~ Calibration standards were prepared in 1 mL of pooled human urine spiked with 25 pL of the stock solution of AFB2. To all urine samples (unknowns, QCs, blanks, and stan- dards) an equal amount of 85:15 MeOH/H20 was added. For ex- ample, the highest concentration standard was made by mixing (per 1 mL of urine) 250 pL of the 1 ng/pL aflatoxin mixture, and 25 pL of the 1 ng/pL internal standard for a total volume of 1.275 mL. To the other samples the same amount of urine and internal standard were used, but with varying amounts of aflatoxin standard and 85:15 MeOH/H20 while maintaining a total volume of 1.275 mL. The final concentrations of the urine standards were: 0.392, 0.784, 3.92, 7.84, 19.6, 58.8, 118, and 196 ng/mL for the calibration standards, 1.96 and 157 ng/mL for the QC low and high, respectively, and the internal standard concentration in all samples was 19.6 ng/mL.

Extraction Urine (1 mL) and water (1 mL) were added to glass tubes (10

x 75 ram), and empty tubes of the same size (for elution) were

AFBI AJ"~

^F01 AF(J2

AI~ AFp I

Figure I. Structures of the four parent aflatoxins and the two metabolites of AFBI monitored in this study.

inserted into the Gilson 215 Liquid Handler (Middleton, WI) for automated extraction. Custom-made immunoaffinity columns, (3-mL barrel, 400 ng aflatoxin equivalent binding capacity, Vicam, Watertown, MA) were used for the extraction. The buffer was discarded followed by rinsing the column twice with water, leaving a small amount of water on top of the resin. The columns were then conditioned with water (2 x 2 mL). After the diluted urine sample was loaded, the column was washed with water (2 x 2 mL), followed by an air push (3 s) using the Gilson solenoid valve. The analytes were eluted from the columns with 85:15 MeOH/H20 with 1% (v/v) formic acid (2 x 0.5 mL), followed by an air push (30 s). The extract was then transferred to an autosampler vial for LC-MS-MS analysis.

LC-MS--MS Chromatography was performed with an Agilent 1100 HPLC

(Wilmington, DE) equipped with a 3-pro, 2.0- x 150-ram phenyl-hexyl column (Phenomenex, Torrance, CA) at 50~ Injections of the extract (1 pL) were made on the HPLC using a mobile phase consisting of H20 (0.1% formic acid) (solvent A) and acetonitrile (0.1% formic acid) (solvent B). The mobile phase gradient is given in Table I.

Samples were analyzed by positive ion electrospray-MS-MS spectrometry operating in multiple reaction monitoring (MRM) mode on an API 4000 LC-MS-MS system (Applied Biosystems, Foster City, CA). The MS settings are listed in Table II. Individual compound specific parameters (i.e., declus- tering potentials, entrance potentials, and collision cell exit po- tentials) were optimized for each analyte.

Data analysis and recovery The product ion abundances of the analyte and internal stan-

dard were used to calculate analyte/internal standard ratios for quantitation. Linear regression analysis with "l/x" weighting was used for curve fitting. All data processing was performed

Table I. Instrument Parameters for LC

Time (min) Rate (mL/min) %B

0.00 0.325 30 10.00 0.325 30 10.10 0.325 95 11.50 0.325 95 11.51 0.325 30 15.50 0.325 30

Table II. Instrument Parameters for MS

Analyte Precursor CE Product

AFP1 299 33.0 271 AFM1 329 33.0 273 AFG 2 331 35.0 313 AFB 2 315 37.0 287 AFG 1 329 39.0 243 AFB 1 313 33.7 285

151

automatically using the Analyst 1.4 software (Applied Biosys- tems). Extraction efficiencies were calculated as a percentage of the ratio of extracted analyte peak area and the non-extracted standard peak area. Four extractions over a period of two days were performed for this study, with all at a concentration of 7.84 ng/mL. Four injections of non-extracted standard at an equal concentration were also made. The peak areas for the extracted and non-extracted samples were averaged and this average was used to calculate the ratio for each analyte.

Animal study Urine samples were obtained from two male F344 rats

(173-176 g body weight). AFB1 (91 pg/kg body weight) or the vehicle (dimethylsulfoxide) were administered by intraperi- toneal injection (150 IJL) on two consecutive days, and the rats were housed in metabolic cages. Urine was collected for ap- proximately 18 h after the second dose and stored at -20~ Urine aliquots (1 mL) were treated with 250 pL of 85:15 MeOH/H20, and 1 mL of this mixture was then extracted. The animal study was conducted in accordance with Johns Hopkins University's Animal Care and Use Committee requirements, which comply with the National Research Council's Guide for the Care and Use of Laboratory Animals.

Results and Discussion

The parent aflatoxins AFB], AFB2, AFG], and AFG2, and two phase I oxidative metabolites of AFB], AFM], and AFP] (9), were selected for analysis. Both AFP1 and AFM] are excreted in human urine (10) and studies have demonstrated that AFM] is the most abundant AFB1 metabolite found in the urine of rats and humans (11). Furthermore, research involving AFM1 has shown that urinary AFM1 levels reflect exposure in humans (12). Metabolites of AFB2, AFG1, and AFG2 were not included in this study because these three compounds are generally not observed in the absence of AFB1, and AFB1 is the most oc- current aflatoxin found in food (13).

Urine (vs. blood or serum) was chosen as the sample matrix primarily due to its ease of collection. If victims are in rea- sonably stable condition, they can provide a urine sample without the assistance of a medical professional. Minimizing the workload of hospital staff is critical in a scenario such as a terrorist attack when hospitals will likely be overwhelmed.

AFB2 was chosen as a chemical analogue internal standard because it behaves similarly to the other aflatoxins both in the immunoaffinity column (IAC) and in the analytical method. In addition, AFB2 is chromatographically well-resolved from the other analytes and less toxic than AFB1 and AFG]. Chemical analogue internal standardization was employed for two rea- sons. First, isotopically labeled standards were only available for AFB2 and were prohibitively expensive (~ $2000 for 8.3 IJg AFB2 3H). Second, the upper limit of linearity (ULOL) was de- termined by occupying the available binding sites of antibody in the column. The use of isotopically labeled internal standards for each aflatoxin would result in more aflatoxin being added to the immunoaffinity column, which would lower the ULOL and,

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Journal of Analytical Toxicology, Vol. 31, April 2007

therefore, decrease the dynamic range of the method. If AFB2 were in a real world sample, a t-test could be per-

formed to determine if the internal standard peak areas in un- known samples are significantly higher than the mean AFB2 peak area in the calibration standards. If this were the case, then because all of the compounds of interest are equally suit- able as chemical analogue internal standards, an aflatoxin not present in the sample would be added to a separate aliquot of unknown and then re-extracted. A second alternative would be to measure the concentration of AFB~ by the method of stan- dard additions. However, both of these methods would be dif- ficult if the concentration of AFB2 was at or near the limit of detection (LOD), so a third alternative, analyzing each un- known in duplicate (one with internal standard, one without), could be employed.

Immunoaffinity extraction of aflatoxins in various matrices has been reviewed (14) and involves non-covalent binding of the toxins to monoclonal antibodies in aqueous environment followed by release upon denaturing of the antibodies using high organic content solvents. After a comparison with C18 SPE in this laboratory, the immunoaffinity method was chosen because of its increased recovery, selectivity, and cleanliness of extracts. Because the extraction columns were originally man- ufactured for food analysis, the antibody used was designed to target only the parent aflatoxins; however, because of the struc- tural similarities between the parent and metabolites, the metabolites were efficiently extracted as well. For this reason, the columns could readily be used for clinical samples.

Table III. Extraction Efficiency for all Five Analytes Measured at 7.84 ng/mL in Urine (n = 4)

Analyte Extraction Efficiency (%)

AFB 1 87.4 + 8.2 AFM1 81.9 + 3.0 AFP~ 80.3 _+ 4.9 AFG1 93.2 _+ 8.5 AFG 2 84.5 _+ 8.8

2~4

2~e4

2~4

b4

t 4e4

60WO

4~00

2~00

AFM~

] AFBI I AFG' I

AFP I | AFG:

s '

:~ :~e: : : : : : : : : : : : : : : : : : : : : :fa :;6 :g/~ u~e ::tra:

Journal of Analytical Toxicology, Vol. 3t, April 2007

Automated extraction was utilized as a means to increase sample throughput. The extraction took 1.67 min/sample, cor- responding to 36 samples/h. The efficiency of the automated extraction was measured at a concentration of 7.84 ng/mL in urine. The extraction efficiencies for each of the five com- pounds are shown in Table III. The results ranged from 80 to 93% and were in agreement with manufacturer specifications, which were defined for food matrices.

The best chromatographic resolution was obtained using a phenyl-hexyl column. Figure 2 depicts the separation of all six aflatoxins used in this study extracted fromhuman urine at a concentration of 19.6 ng/mL. The MS-MS fragmentations of the aflatoxins are shown in Figure 3. It can be seen that both B aflatoxins and the AFB1 metabolites fragment by losing the carbonyl on the cylcopentanone group, and that AFMt further breaks down by losing the two adjacent CH2 groups on the

/31~ ,114,

'9 A '

~Oo4i , J~ i

Slt~I ,,3~+

+t.. ,,,++ '."+ .. + t , - i

t,.,++ I: , + ,+.++ ,~~ ,,~,+ I, ,m,,j

+ mPo.~ + "-0+ ~,, T ,+:

11 ...................... J.._+w.'l. ................................ ~ ............................. " 1+

D

m, l l

~,., ~+..++. m. .~ ~, m . - mo m-++. . .+ m~"

iil I~I.O!

-+I ii i' | + ,+ ++++., + ++T+ ,++ +.+ , . ++, + + +_.++...++,J++,. ++,. +.

im~M)

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:=i/ ! i= t:.: l / " ,.,,-i I

10~oJ I

=i / , '~t / ",

~+-m+. ~+++- +.+ ++..~.....-..++--,++-- +...+++,+.+.+.++..... +..++.+.++..+ +..++ . ,~(Nm)

,-! C .... + F J ,.!

9 [ 11~4 i

|+ i ! '-~ - ,-i ! -~ Mm.~

1~01 i ~ mr , 1~118 ~ i I 1 i i~ o ml _ ~ ___

Figure 3. MS-MS fragmentation spectra for each aflatoxin with arrows indicating the precursor ion: AFB; (A), AFB+ (B), AFG1 (C), AFG+ (D), AFM1 (E), and AFP; (F).

153

ring. The G aflatoxins differ by containing a lactone group in place of the pentanone ring structure. These compounds frag- ment by the loss of water and the further loss of the outer car- bonyl group and adjacent portion of the ring, as well as the loss of H2. The fragmentation patterns suggest that the site of pro- tonation for each aflatoxin was the inner carbonyl group. These fragmentation patterns were based upon interpretation of the MS-MS spectra because isotopically labeled experiments were not performed for reasons stated previously.

The instrument LOD (S/N = 3:1) for each compound is given in Table IV. LOD values were reported in femtograms (fg) on column to avoid ambiguity inherent in using units of concen- tration such as parts per billion (ppb). This ambiguity made it difficult to determine a relative LOD in papers where the per- tinent information to calculate the LOD on column was not in- cluded (15,16). By providing the LOD as fg on column, the values reported in this manuscript are independent of the volume of urine extracted and/or injection volume, and they re- flect the amount of sample that was detected by this method. Table IV shows the LOD for each analyte in terms of grams on- column and in moles. An LOD of 100 fg is equivalent to a 1-~L injection of non-extracted standard at 100 fg/l~L. These values meet the goal of developing a sensitive method as these detec- tion limits are more than 10 times lower than those previously reported (17,18) for aflatoxins in urine. The limit of quantita- tion (LOQ) (S/N = 10:1) was calculated in urine using the same procedure stated previously. The LOQ was determined to be 392 fg on-column for all analytes.

A wide dynamic range is needed in the analysis of chemical

Table IV. LOD for Each Analyte Defined as a Signal-to- Noise of 3:1

Analyte Femtograms on Column Attomoles

AFP~ 50 168 AFM~ 100 305 AFG2 100 302 AFG~ 100 305 AFBI 50 160

i LIk 5

Concentration (n~/raL) Figure 4. An eight-point calibration plot from 0.392 to 196 ng/mL in urine using linear regression with "l/x" weighting.

Journal of Analytical Toxicology, Vol. 31, April 2007

warfare agents for two reasons. First, because there are no studies in the literature showing the range of concentrations in a victim exposed to weaponized aflatoxin, having a dynamic range of nearly three orders of magnitude increases the likeli- hood that this method will cover relevant concentrations. Second, the exposure of victims after an event will not be uni- form because of their different proximities to the attack epi- center. To achieve this goal, custom-made extraction columns with a larger bed size were employed. The dynamic range of the method was from the LOQ at 0.392-196 pg on-column for each analyte. Procedures that required time consuming con- centration steps, which may allow for the detection of lower aflatoxin levels, were avoided to increase assay throughput [i.e., 1 mL of urine was extracted into 1 mL of MeOH/H20 with 1% (v/v) formic acid]. Figure 4 shows a calibration curve for all five toxins in human urine. The dynamic range pre- sented here meets the aforementioned goal of covering a wider range than those previously reported (8,17).

As seen in Figure 4, a slight deviation from linearity begins to occur at the highest point. This is due to the large aflatoxin/an- tibody ratio at this concentration. This was examined by ana- lyzing non-extracted standards that showed increased linearity at this same concentration and at even higher concentrations (data not presented), which verified that our ULOL was dictated by the binding capacity of the extraction column and not by the instrument. At concentrations above the ULOL, the number of

AFG+ QCL A

A 9 9

8 9 9

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . & . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

AFGI QCH B

+,,~ i ,

w w

Figure 5. Quality control plots for AFG~, QC Low at 1.96 ng/mk (A)and QC High at 157 ng/mL (g). The central line represents the mean, the dashed line is two standard deviations, and the outer line is three stan- dard deviations from the mean.

154

Journal of Analytical Toxicology, Vol. 31, April 2007

binding sites became a limiting factor and some unbound aria- toxin was removed during the wash step of the extraction. This was an important consideration when determining the ULOL of an extraction method using immunoaffinity columns.

The method was validated by analyzing a calibration curve, two quality controls (low and high), and a urine blank spiked with internal standard. This experiment was repeated 20 times over a period of 12 days with no more than 2 sets being analyzed in a single day. Eight analysts conducted the experiments

Table V. Validation Results Depicting the Precision and Accuracy of the Method.

Lower Upper Concentration Mean % 95% Limit 95% Limit

Analyle (ng/mL) (ng/mL) Accuracy (ng/mL) (ng/mL)

AFB 1 1.96 2.01 103 1.95 2.08 AFB 1 157 153 97.5 149 157 AFM1 1.96 2.01 102 1.95 2.07 AFM~ 157 152 97.0 148 156 AFG1 1.96 2.07 106 2.02 2.13 AFG~ 157 152 97.0 150 155 AFP1 1.96 2.03 104 1.97 2.09 AFP1 157 151 96.5 147 155 AFG2 1.96 2.06 105 2.02 2.11 AFG2 157 146 93.5 144 150

~k,~ i i

i f,*sl

AFMI A

AFF

AFB 2

]~ AFB,

1re(m|)

AFB 2

1o . 12 S3 14 IS

B

1 = 3 4 s" T ; * ~o . ,2 1] 14 is ' r im* ( id l )

Figure 6. Chromatograms from the animal study, dosed rat (A) and con- trol rat (B).

during the 12-day period. Linear regression with "l/x" weighting was used for each analyte to account for het- eroscedasticity in the data. The calibration standards were an- alyzed in a random order and the curves for each aflatoxin had an average correlation coefficient > 0.995. The quality of the method was represented by quality control low and high plots of AFG1 (Figure 5). Similar results were obtained for the four other toxins. The results of the accuracy and precision of the method for each analyte are shown in Table V. The range of the

percent accuracy of the means for all five ana- lytes was from 97.0 to 105.6%, and the highest % relative standard deviation (RSD) found was 6.67%. In all but one case, the mean was s one standard deviation away from the true value. No significant contributions from carryover

% RSD were seen in the blank samples. After validation, the method was further

6.67 tested by analyzing the urine of an AFB1 ex- 5.18 posed rat. The dose administered to the rat (91 6.59 IJg/kg body weight) was well below the LDs0 5.58 for rats and corresponded to a dose of several 5.68 milligrams for a human adult. A dose of 2-6 3.o4 mg/day was observed during an outbreak of 6.30 aflatoxicosis in western India (19), and similar 5.16 amounts were consumed in a recent outbreak 4.79 in Kenya (20). It was expected that roughly 5.07

equal amounts of the two metabolites AFM] and AFP1, and a small amount of unmetabo-

lized ARB1, would be excreted. No aflatoxins other than the in- ternal standard were detected in the negative control rat urine sample. AFBI, AFMI, and AFP] were detected in urine from the AFBl-dosed rat at 1.38, 48.8, and 41.4 n~mL, respectively; 4 mL of urine was collected and indicated total excretion of 5.52, 195.2, and 165.6 ng, respectively, during the 18-h period. The creatinine levels of the two urines were not measured; thus, no comparison of the metabolite concentrations with literature values were made. However, relative to AFP], the amount of AFM 1 was more abundant, which is consistent with previous studies of AFB1 exposure in rats (11,21). Figure 6 shows the chromatograms for the negative control and AFB1 dosed rat urine samples.

It was also expected that other metabolites of aflatoxin would be present in the dosed rat urine. Precursor and product ion masses of other AFB] metabolites (18) were used to build a sep- arate MP~ method to detect the presence of these compounds. Metabolites such as AFB-diol and AFQ] were found. The presence of AFQI was further confirmed by matching the retention time with a chromatogram obtained previously when AFQ] was com- mercially available (data not presented). This method was suit- able for monitoring other metabolites on a qualitative level, but quantitative analysis was difficult because AFM] and AFP 1 are the only commercially available AFB] metabolite standards.

Conclusions

The LC-MS-MS method described uses less urine and has lower limits of detection than previously reported methods.

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Journal of Analytical Toxicology, Vol. 31, April 2007

Other advantages of this approach are the specificity of both immunoaffinity extraction and tandem MS and no require- ment for derivatization. The dynamic range of 0.392-196 pg on column, combined with an automated extraction, yield rapid results over a wide range of exposure. This method is well suited to aid forensic and public health laboratories during the investigation of a terrorist attack by providing confirmation of military or civilian exposure to weaponized aflatoxin. In ad- dition, the method presented here can be utilized to diagnose aflatoxicosis (22) caused by consumption of a food supply con- taminated intentionally by the hands of terrorists or acciden- tally during an outbreak (19,23,24).

Acknowledgments

The authors would like to thank Erin Carson, Mike Martin, Chris Nixon, Jeff Snow, Shane Wyatt, and Jessica Zuckschw- erdt, who contributed to the validation process, and Janet Pruitt for the insightful discussions. The authors would also like to thank Dr. Torn York for his comments to the manuscript. Funding for the analytical work (R.E., EC., D.Z., and T.C.) was funded by the Centers for Disease Control and Prevention grant # U90/CCU317014. The animal study (P.S. and J.G.) work was funded by the NIH grants # P01 ES06052 and #P30 ES03819.

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Manuscript received October 4, 2006; revision received November 27, 2006.

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