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TOF-MS
a n a l y t i c a c h i m i c a a c t a 6 1 8 ( 2 0 0 8 ) 86–93
available at www.sciencedirect.com
journal homepage: www.elsevier.com/locate/aca
Confirmatory analysis of Trenbolone using accurate mass measurement with LC/TOF-MS M.H. Blokland a,∗ , P.W. Zoontjes a , S.S. Sterk a , R.W. Stephany a , J. Zweigenbaum b , L.A. van Ginkel a a b
National Institute of Public Health and the Environment (RIVM), Bilthoven, The Netherlands Agilent Technologies, Wilmington, DE, USA
a r t i c l e
i n f o
a b s t r a c t
Article history:
The use of accurate mass measurement as a confirmation tool is examined on a TOF-MS
Received 3 December 2007
and compared with confirmation using a triple quadrupole mass spectrometer (QqQ-MS).
Received in revised form
Confirmation of the identity of a substance using mass-spectrometric detection has been
12 April 2008
described. However, the use of accurate mass measurement for confirmatory analysis has
Accepted 14 April 2008
not been taken into account. In this study, criteria for confirmation with accurate mass are
Published on line 24 April 2008
proposed and feasibility is demonstrated. Mass accuracy better than 3 ppm of the quasimolecular ion and a fragment and their relative ratios determined with LC/TOF-MS are
Keywords:
compared to the criteria of two transition ions and their ratio of LC/QqQ-MS. The results
Accurate mass measurement
show that these criteria can be met for Trenbolone in samples of bovine urine and that single
Exact mass
MS accurate mass measurement is comparable to nominal mass MS/MS for confirmation.
Time of Flight Mass Spectrometry
The increase in popularity and availability of LC/TOF-MS instruments and the ease, of which
Triple Quadrupole mass
exact masses can be measured, make it important to formulate criteria for this type of
spectrometry
instrumentation. It is shown in this study that accurate mass measurement can be used for
Confirmation
confirmatory analysis. However, more experiments need to be conducted to demonstrate the applicability of accurate mass measurement in general for residue analysis. © 2008 Elsevier B.V. All rights reserved.
1.
Introduction
In residue analysis, mass spectrometry is widely used to identify and confirm regulated and banned substances in food products, the environment, and toxicology. To identify these substances, targeted analysis strategies are typically used. In practice, samples are typically screened using a list of compounds. After identification of a compound by the screening process, confirmation is needed. Confirmation entails the utilization of an analytical method with high selectivity, usually with different characteristics than the screening method, and with direct comparison to an authentic standard. The ultimate
goal of confirmation is unambiguous identification or, put another way, the exclusion of all other possible compounds [1]. The formulation of specific criteria and use of identification points is widely adapted for compound confirmation using chromatography combined with mass spectrometry [2]. This has been written into European Union legislation and, depending on the purpose of the analysis and legal status of the analyte, it is necessary to collect three or four identification points for confirmation [3]. These points are obtained by measurement of at least two ions (in the case of MS/MS or high-resolution MS) of which their ratios are calculated and compared with those obtained from authentic standards
∗ Corresponding author at: National Institute of Public Health and the Environment (RIVM), P.O. Box 1, 3720BA Bilthoven, The Netherlands. Tel.: +31 302743966. E-mail address: [email protected] (M.H. Blokland). 0003-2670/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.aca.2008.04.040
a n a l y t i c a c h i m i c a a c t a 6 1 8 ( 2 0 0 8 ) 86–93
under the same analytical conditions. When these ratios are within predefined limits and the retention time of these ions matches the analytical standard, the required points are obtained, thus providing confirmation [4]. In view of the low concentrations in real samples at which confirmation of, e.g. growth promoting agents is required (for example biological materials obtained from cattle), the criteria for confirmation analysis using mass spectrometry are most important. The MS-identification criteria (identification points or IP) are described for different mass analyzers and depend on the method of fragmentation (tandem MS vs. single MS) and the resolution at which the ions are detected. The criteria specify that a mass spectrometer must be used in combination with a separation technique such as gas chromatography (GC) or liquid chromatography (LC). For LC/MS1 these criteria limit laboratories to certain types of instruments and because of the limitation of these instruments, it is virtually impossible to determine whether there are other than the targeted compounds present. To overcome this problem and to perform non-targeted analyses (compounds of interest but not on the screening list), alternatively TOF instruments can be used. However, TOF instruments are, according to current guidelines, treated equally as single quadrupole instruments due to their limited resolution. In practice, this means that after a non-compliant finding the samples have to be re-analysed on a tandem mass spectrometer. Resolution and mass accuracy are the two critical factors when different type of MS principles are compared and evaluated. Resolution is the degree of separation between two adjacent ions in the mass spectrum that can be distinguished at a given mass. A typical TOF mass analyzer can resolve mass differences of a few hundredths of a mass unit to a few tenths of a mass unit (e.g. 0.025–0.25 u). Mass accuracy is the difference between the theoretical (calculated) mass of a compound and the mass measured by the mass spectrometer. Mass accuracy/error can be expressed in either parts-permillion (ppm) or milli-mass units (mmu). A typical TOF can determine the accurate mass with a error of 3 ppm or better. The use of accurate mass measurement is not taken into account in current confirmation guidelines [3] and more importantly the degree of mass accuracy needed for confirmation is not specified. The closest criterion for accurate mass measurement is the high-resolution criterion defined as: In high-resolution mass spectrometry (HRMS), the resolution shall typically be greater than 10,000 for the entire mass range at 10% valley. It may be assumed that in performing highresolution mass spectrometry the MS is properly tuned and calibrated, and is capable to analyze samples within a few ppm of the theoretical exact mass. However, because a criterion for mass accuracy is not defined in the Commission Decision 2002/657/EC or other guidelines [5] it cannot be assumed that a measurement is accurate to a sufficient level no matter how great the mass resolution. Moreover, when
1
Because of the nature of fragmentation in LC/MS (low energy collision induced dissociation) for a significant number of compounds, it is impossible to obtain the four ions required with single MS and/or meet the sensitivity requirement.
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making high-resolution measurements in selected ion monitoring (SIM), an abundant co-eluting compound with a mass close to the recorded SIM mass, can result in an erroneous signal at the measured SIM mass. Criteria for accurate mass measurements were described in 1983 [6] and were included in Commission Decision 87/410/EEC [7]. These early criteria were based on mass measurements with accuracy expressed in milli Dalton error calculated from the exact mass. Unfortunately, these criteria “disappeared” in 1993 from Commission Decision 93/256/EC [8] and were not included in 2002/657/EC [3]. The use of accurate mass measurement for confirmatory analysis, however, was explored and used [9]. It was stated that mass errors below 2 mDa were generally accepted as accurate mass measurements [10]. In a recent report, accurate mass measurement was described as a valuable tool for confirmation and indicated the specificity gains from 5 ppm error to 2 ppm [11]. However, no recommendation on what would be an acceptable tolerance was given. In a recent paper [12] criteria were proposed for confirmation analysis using accurate mass measurements: for TOF instruments (mass resolving power (FWHM) ≥10,000) 1.5 point/ion or product ion with at least one ratio. As mass accuracy criterion ≤5 mDa was proposed. However, when mDa error is expressed in the commonly used ppm error [13,14] an accurate mass measurement with an error of 5 mDa for small molecules, for example 300 g mol−1 , translates to a mass accuracy of 16.6 ppm, and at mass 1000 g mol−1 it is 5 ppm. Modern instruments can do better than 3 ppm for masses below 1000 g mol−1 . For small molecules the proposed criteria of 5 mDa will generate falsely identified compounds. The use of a more strict criterion in ppm error for molecules with a mass less than 1000 gains much more selectivity than the use of a mDa error and, as we will show, may be more important for confirmation than resolving power. An exploration of the use of accurate mass measurement to gain identification points is important. The measurement of only one exact mass will not be sufficient to confirm the identity. With a typical mass accuracy of 3 ppm, several elemental compositions usually remain possible. Moreover, the measurement of a single exact mass gives no information about the structure of a compound. Therefore, measuring a fragment ion within 3 ppm of the exact mass along with the quasi-molecular ion is necessary. This gives structural information about the compound measured and correlates the proposed molecular formula of the two ions (the fragment’s ion formula must be a subset of the quasi-molecular ion formula). We and others propose that this combined with the ion-ratio criteria as already defined in CD 2002/657/EC and matching chromatographic retention time would constitute confirmation [12,15]. This work is novel in that it reports the use of a single TOFMS with the capability of routinely measuring highly accurate masses for confirmation using two ions, the quasi-molecular ion and a resulting fragment generated by in-source collision induced dissociation. This is the first report in which the ion ratios obtained from these measurements are compared with those obtained with LC/QqQ-MS. In addition, good chromatographic separation and matching retention times with that of authentic standards remains an important criterion for confirmation.
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This study investigates the use of accurate mass measurements with an LC/TOF-MS, using the above described criteria for confirmation of the identity of a compound and compares the results with those obtained by QqQ-MS/MS measurements. The relationship between mass accuracy, ion ratios, and retention-times is described and evaluated with respect to confirmation and its ultimate goal of unambiguous identification.
2.
Experimental
2.1.
Materials
All solvents used were of analytical grade or better. Deionized water was obtained from a Milli-Q purification system. The compounds 17␣- and -Trenbolone and deuterated 17Trenbolone-D3 were obtained from the “Bank of Reference Standards” (RIVM, the Netherlands). Immunoaffinity columns (IAC) were obtained from Randox (Ireland).
2.2.
Hydrolysis of urine
A test portion of 5.0 mL bovine urine was pipetted into a 50mL plastic centrifuge tube; internal standard, 50 L of 0.1 ng 17-Trenbolone-D3 L−1 , was added and mixed. The pH was adjusted to 5.2 with 2 mL acetate–buffer (2 mol L−1 ). To deconjugate glucuronide and sulfate conjugates, 75 L suc d’Helix Pomatia was added and the tube was vortexed. The mixture was incubated for 3 h at 37 ◦ C.
2.3.
Liquid/liquid extraction (LLE)
LLE was performed with 10 mL of hexane/butanol (80/20, v/v%). After centrifugation (2 min at 3000 × g), the top layer was transferred to a glass tube and evaporated to dryness under a stream of nitrogen at 55 ◦ C. The dry residue was dissolved in 250 L of ethanol after which 5 mL of water was added.
2.4.
Immunoaffinity chromatography (IAC)
The IAC-Trenbolone column was conditioned by washing twice with 5 mL of wash-buffer (provided by Randox). The total sample extract was applied to the IAC-column. After sample application the column was washed twice with 5 mL of buffer and with 5 mL of water, and eluted with 5 mL of 70/30 (v/v) ethanol/water. The eluate was evaporated to dryness under a stream of nitrogen at 55 ◦ C. The sample was redissolved in 500 L of ethanol and transferred to a 2 mL vial. The ethanol was evaporated to dryness at 50 ◦ C and 100 L acetonitrile/water (40/60, v/v) was added. The vial was vortexed for 30 s. The mixture was transferred into an insert and the vial was capped, ready for LC/MS analysis.
2.5.
LC/TOF-MS
LC/TOF-MS measurements were performed on an Agilent 6210 LC/TOF-MS coupled via an APCI interface to an Agilent LC1200 SL binary system with column oven. The LC column used for the TOF experiments was a Zorbax Rapid Resolution C18
(2.1 mm × 50 mm, d.f. = 1.8 m). Mobile phase A consisted of 10/90 (v/v) acetonitrile/water, mobile phase B of 90/10 (v/v) acetonitrile/water. The gradient used was linear, started at 40% A and progressed to 90% B in 10 min. After 10 min the mobile phase was kept for 2 min at 90% B. The column oven was set to 80 ◦ C and the mobile phase flow was set at 0.5 mL min−1 . The injection volume was 40 L. The following parameters were used for the LC/TOF-MS measurement. The gas temperature was 325 ◦ C, nitrogen drying gas was set at 10 L min−1 and the nebulizer pressure was 50 psi. The dielectric capillary exit was 250 V (fragmentor setting) and the skimmer 60 V. The octapole DC1 was set to 35.5 V and octapole RF to 250 V. The instrument was tuned and calibrated with the standard calibration solution provided by the manufacture. During analyses, an internal mass reference solution was added via a T-junction. This reference solution contained two compounds, purine and HP-921, with protonated masses of 121.0509 and 922.0098 respectively, which were used for continuous mass axis calibration. Instrument resolution (FWHM) was approximately 10,000 for mass 922.0098. Both the 17␣- and -Trenbolone were “in-source” fragmented in the high-pressure region between the dielectric capillary exit and the skimmer. The measured ions of 17␣/Trenbolone were the protonated molecule m/z 271.16926 amu and the loss of water at m/z 253.15869.
2.6.
LC/MSMS
LC/QqQ-MS measurements were performed on a Waters Micromass Ultima coupled via an ESI interface to a Waters 2695 LC system with column oven. The LC column used for the MSMS experiments was an Alltech Alltima C18 (2.1 mm × 150 mm, d.f. = 5 M). Mobile phase A consisted of 10/90 (v/v) methanol/water, mobile phase B of 90/10 (v/v) methanol/water. The gradient used was linear, started at 50% A and progressed to 80% B in 10 min. After 11 min the mobile phase was kept for 4 min at 100% B and finally returned in 0.1 min to its initial conditions. Mobile phase flow was set at 0.3 mL min−1 , the column oven to 40 ◦ C. The following parameters were used for the LC/MSMS measurement. The electrospray capillary was at 3.2 kV, the cone voltage was 50 V and the collision energy was set at 16.0 eV, argon was used as collision gas at 2.6 × 10−3 mbar. The ion source temperature was 110 ◦ C and the desolvation temperature was 350 ◦ C. Nitrogen was used as nebulizer gas, cone gas flow 113 L h−1 and desolvation gas flow 637 L h−1 . Data were acquired in the multiple reaction monitoring (MRM) mode. The precursor ion for 17␣/-Trenbolone m/z 271, two product ions m/z 199 and m/z 253, the precursor ion for 17-Trenbolone-D3 m/z 274 and one product ion m/z 256 were monitored. The dwell time was set at 100 ms for each.
2.7.
Experiments
2.7.1.
LC/TOF-MS
The exact masses of 17␣/-Trenbolone and 17-TrenboloneD3 were measured at three different levels close to the detection limit of the instrument (1–0.5–0.1 ng on column). To test the capabilities of the LC/TOF-MS for confirmation in samples of bovine urine, the accurate masses of the selected
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ions were determined in 20 different spiked samples at 1 and at 5 g L−1 respectively. After sample preparation a concentration of 1 g L−1 corresponds to the recommended value for screening and confirmation previous know as the minimum required performance limit (MRPL) for this analyte matrix combination. The accurate masses were determined by background subtraction of the signal in front of the peak. The ratios were calculated according CD 2002/657/EC.
2.7.2.
LC/MSMS
The same 20 samples of bovine urine, spiked at 1 g L−1 of 17␣and -Trenbolone and 17-Trenbolone-D3 were analysed with LC/MSMS. For confirmation, the ratios of the two product ions were calculated according to CD 2002/657/EC.
3.
Results and discussion
Using the IP system that has been implemented for confirmation of banned substances in animal and animal products is an appropriate reference for proposing accurate mass criteria. The criteria should include a tolerance for mass at better than 3 ppm; a tolerance for ion ratios, and thus at least two ions to be measured; and a requirement and tolerance for matching chromatographic retention time with a reference compound. An understanding of the need for a strict mass tolerance can be better obtained by taking the protonated exact mass of Trenbolone (C18 H23 O2 ) at m/z 271.1693. Determination of the molecular formula at this mass within 3 to 15 ppm for only the elements C,H,O will only yield C18 H22 O2 . However, this does not eliminate the possibility of a false non-compliant. If the common elements C,H,O,N, and S were included the possibilities increase to eight different molecular formulas at 15 ppm as shown in Table 1. At 3 ppm error the possible formulas are reduced to three. Although the fragment ion from water loss is not considered a unique ion, it is the major fragment of Trenbolone and indicative of a structural characteristic of the molecule. If the same criterion is applied to this fragment ion of Trenbolone, six formulas are possible as shown in Table 2. Of the six formulas, five correlate to formulas of the parent ion, m/z 271.1692, at a tolerance of 15 ppm mass error. At 3 ppm only two formulas are subsets of the parent compound and one of these two, C3 H17 N12 O2 , does not likely represent an actual chemical. Since, this formula is searched on the extensive
Table 1 – Possible formulas for Trenbolone ion (M+H)+ with tolerance to 15 ppm and elements including C,H,N,O,S Formula C18 H23 O2 C3 H19 N12 O3 C11 H23 N6 S C14 H19 N6 C7 H23 N6 O5 C6 H23 N8 O2 S C15 H27 O2 S C13 H23 N2 O4
m/z 271.1692 271.1697 271.1699 271.1665 271.1724 271.1659 271.1726 271.1652
Error (ppm) 0.0 −1.8 −2.5 9.9 −11.7 12.3 −12.4 14.8
Table 2 – Possible formulas for Trenbolone fragment ion (M+H-H2 O)+ with tolerance to 15 ppm and elements including C,H,N,O,S Formula C18 H21 O C3 H17 N12 O2 C10 H25 N2 O3 S C7 H21 N6 O4 C6 H21 N8 OS C15 H25 OS
m/z 253.1586 253.1591 253.158 253.1618 253.1553 253.162
Error (ppm) 0.0 −2.0 2.6 −12.6 13.2 −13.3
Internet accessible database, PubChem [16], no chemicals are found. This leaves, at 3 ppm error, only the correct formula for Trenbolone and demonstrates that the mass tolerance criteria needs to be restricted to this level to obtain the selectivity needed for confirmation. Having the correct molecular formula does not constitute confirmation in the sense of unambiguous identification. If the formula of the molecule, C18 H22 O2 , (obtained with an accuracy of 3 ppm for the quasi-molecular ion and a fragment) was searched in the same database, PubChem, 487 possible compounds are obtained. However, considering that phenolic OH on a molecule will not yield a water loss fragment and that typically small alkyl OH will readily loose the alcohol and not water, we can reduce the number of possible compounds from 487 to 59 containing an OH that would readily loose water. Of those 59 in the database, three are listed as Trenbolone, 17␣-Trenbolone, and 17-Trenbolone leaving 56 other compounds that could generate a false non-compliant. This approach towards showing specificity is not unique as Sphone demonstrated that the 3 ion criterion with single MS MIM was effective in searching a large library [17]. When Sphone reported his results, the library he used (MSSS) contained over 30,000 compounds. The database used here for searching unique molecular formulas contains over 10,000,000 compounds. This search shows how many possible compounds could interfere and highlights the importance of good chromatographic separation and the need to measure relative ion ratios to attain an unambiguous identification for confirmation. A similar search of mono-isotopic masses from 269.8–270.3 (nominal mass measured with triple quadrupole instruments) yields 20,878 compounds in this database. Because LC/MS/MS has become the standard for confirmation in residue analysis it becomes evident that the need for good chromatographic separation and measurement of ion ratios is just as important using this technology. Also with nominal mass triple quadrupole instruments set to unit resolution, all isotopic information is lost allowing inclusion of elements such as the halogens as possible interferences. The first experiment examined whether the identity of 17␣/-Trenbolone could be confirmed based on the proposed accurate mass criterion for LC/TOF-MS. For this purpose, low concentrations of the compounds in pure solvent were analyzed near the expected limit of detection of the analytical method. For the two ions selected, the accurate masses were measured (criterion ≤3 ppm deviation). To determine the number of identification points acquired, the same allocation as prescribed for high-resolution mass spectrometry was followed [3]. Each measured accurate mass yielded 2 points
3.3 19.2 5 0 1.0 14.6 10 4 0.5 10 8 17-Trenbolone 271.1693 253.1514
14.2
7.8 18.2 5 2 1.3 19.2 10 6 0.5 19.5 10 10 17␣-Trenbolone 271.1693 253.1587
Average ratio (%) Number < 3 ppm Number < 3 ppm Average ratio (%)
Uncertainty (S.D.%) Number < 3 ppm
Average ratio (%)
Uncertainty (S.D.%)
0.1 ng (n = 10) 0.5 ng (n = 10) 1.0 ng (n = 10)
if it was within 3 ppm of the theoretical mass. Using these standards a ratio criterion must be established. Thus, the repeatability of the ratio is very important. It is on the basis of the average value of the ratio for the standards that unknown signals are evaluated. Therefore, the uncertainty of the average value as determined for the standard substance must be small in comparison with the tolerance interval allowed. In Table 3 an overview of the results for the standards is shown. From Table 3 it is observed that at 1 ng on-column it is possible to measure the relative intensity with very good reproducibly. For 17␣-Trenbolone, the most dominant metabolite of Trenbolone, when analyzing samples of urine, all measured masses were within 3 ppm of the theoretical value. In two samples the error for m/z 253 for 17-Trenbolone was larger then 3 ppm. This gives in total 18 successful determinations within 20 tests. At 0.5 ng on-column, however, the mass accuracy was not within 3 ppm for 10 individual measurements out of 40 determinations and at 0.1 ng on-column 28 determinations failed. This failing of confirmation in these cases is due to the low signal intensity of the ions. Therefore, it is concluded that 1 ng on-column is the lower limit for confirmation using the criterion of ≤3 ppm mass measurement error. Furthermore, Table 3 shows that the repeatability of the ratios is acceptable for the standards at 1.0 and 0.5 ng oncolumn, with only small differences between these amounts tested. The repeatability for the standard at 0.1 ng is not as good, and again this can be attributed to the low signal for this concentration of the standard. From the experiment with standards, it can be concluded that reliable confirmation is possible when approximately 1 ng 17␣- or 17-Trenbolone is injected on-column. Based on a minimum recommended value of 1 g L−1 for both compounds and a sample portion of 5 mL, this is sufficient. However, other factors such as matrix interferences could reduce the sensitivity in real samples. For testing of the confirmation capabilities of the LC/TOF-MS, 20 different samples of bovine urine were spiked at 1 g L−1 . In Fig. 1 a typical chromatogram and spectrum are shown for a sample of urine spiked at 1 g L−1 . The loss of water (m/z 271 → 253) is in general not considered a specific loss, however, if the accurate mass of this fragment ion is within the subset of the fragment’s of the quasi-molecular ion, it can be considered as diagnostic. Of course if there are other fragment ions available within the subset of the quasimolecular ion these also should be considered. As can be seen from the chromatogram and spectrum no matrix interferences are visible. Based on these data, it should be possible to confirm the identity at mass concentrations of 1 g L−1 as compared to the standard at 1 ng on-column. The results of the accurate mass measurements and their ion ratios of the spiked urine samples are given in Table 4. For 17␣-Trenbolone, all measured masses for m/z 271 are within 3 ppm of the theoretical values and therefore fulfill the mass accuracy criterion. However, 3 measured masses for m/z 253 deviate by more then 3 ppm and therefore failed the mass accuracy criterion. The ratios of 271/253 are all within the limits of the reference value as given in the 2002/657/EC. Therefore, in total, the identity of 17␣-Trenbolone was confirmed in 17 out of 20 samples. The false compliant rate (<4 IP), therefore, is 15%. For 17-Trenbolone one ion at m/z of 271 and
Uncertainty (S.D.%)
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Table 3 – Results of the accurate mass measurement of 10 injections of the test compounds in pure solvent at three different levels (1, 0.5, 0.1 ng on-column)
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Fig. 1 – (A) LC/TOF-MS chromatogram of 17␣/-Trenbolone. (B) Mass spectrum of 17-Trenbolone, arrows point to the m/z used for confirmation analysis.
three at m/z 253 did not fulfill the accurate mass measurement criterion. Nevertheless, all relative ratios for 17-Trenbolone were within the limits given in CD 2002/657/EC. Therefore, in 20% of these samples a false compliant result (<4 IP) was obtained.
Thus, for confirmation with LC/TOF-MS in 20 samples containing two compounds each, seven instances failed. In those seven cases, the mass deviation was between 3 and 5 ppm for 5 measurements. In the other two cases, the deviation was higher, with a maximum measured deviation of 7.74. This
Table 4 – Determination of the exact mass and ion ratio in 1 g L−1 spiked urine samples 17␣-Trenbolonea (271.169, <3 ppm) −0.81 −0.74 −2.29 −1.99 0.41 0.41 −0.22 −0.44 −0.33 −1.66 −0.44 −0.37 0.55 0.18 0.00 0.96 0.63 0.81 1.81 1.14 a b
17-Trenboloneb (253.158, <3 ppm) 0.16 1.66 −1.22 −2.09 2.61 2.13 −0.71 0.40 1.82 0.95 1.15 2.05 3.87 3.28 2.41 2.77 2.92 2.80 4.15 2.61
Ratio 271/253 = 18.0 18.6 16.0 18.2 16.9 18.3 17.6 18.7 18.8 17.2 19.3 16.8 17.1 17.6 18.1 16.0 18.2 17.4 20.2 18.0 18.9
IP
271.169, <3 ppm ()
253.158, <3 ppm ()
4 4 4 4 4 4 4 4 4 4 4 4 2 2 4 4 4 4 2 4
0.32 −0.33 −5.31 −1.92 0.48 −2.77 −1.00 −2.51 −0.81 1.33 −1.99 −1.88 0.07 0.89 0.77 0.77 −0.92 −0.52 1.51 1.18
−0.48 4.70 −2.84 2.69 2.69 −0.36 2.49 0.79 2.45 −2.53 1.15 0.16 2.29 2.92 0.83 3.00 3.36 1.30 7.74 2.13
Ratio 271/253 = 12.9 12.3 12.5 12.9 14.5 12.9 13.0 13.2 12.7 13.4 13.2 12.7 12.2 12.6 12.9 12.3 12.9 13.1 13.1 13.3 13.4
IP 4 4 2 4 4 4 4 4 4 4 4 4 4 4 4 4 2 4 2 4
17␣-Trenbolone: lower limit 12.6; upper limit 23.4. 17-Trenbolone: lower limit 9.1; upper limit 16.8. Lower and upper limits were calculated according Commission Decision 2002/657 (±30% for ions with intensity between 10 and 20%).
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Table 5 – Results of the ratio for two product ions obtained for samples of spiked bovine urine (1 g L−1 ) on a LC/QqQ-MS 17␣-Trenbolonea 253/199 = 19.0 22.4 21.4 21.4 22.0 24.6 26.6 23.9 23.3 26.4 24.4 24.5 24.7 25.6 25.1 30.4 27.4 27.3 28.3 25.7 21.0 a b
Ratio
17-Trenboloneb Ratio 253/199 = 66.2. 67.5 69.6 75.4 65.6 57.3 63.2 75.6 71.4 71.3 74.5 56.4 54.3 61.6 82.1 67.8 61.7 59.2 83.6 62.8 79.0
17␣-Trenbolone: lower limit 13.3; upper limit 24.7. 17-Trenbolone: lower limit 52.7; upper limit 79.0. . Lower and upper limits were calculated according Commission Decision 2002/657.
is considerably higher than expected based on the measurements made on standards. To check whether the failing of the 3 ppm criterion for confirmation was due to ion-suppression or matrix effects, the same set of urine samples was spiked at 5 g L−1 . In all of these samples the accurate mass criterion was met, and the ratio was well within acceptable tolerance as described in 2002/657/EC (data not shown). To compare the effectiveness of confirmation with a widely accepted technique, the same experiment was repeated on a LC/QqQ-MS system. The results are summarized in Table 5. The criteria for MS/MS specify two product ions are to be measured at the correct chromatographic retention time. The presence of a fragment ion is assessed based on the S/N at the relevant retention time. As a rule, a S/N value of 6–10 is necessary for reliable quantification of the area. This quantification is necessary for determination of the ratio of the two product ions. The two fragment ions each only yield 1.5 IP each. However, the precursor ion is assumed because the first mass filter is set to only let that m/z pass through and thus 1 additional IP is awarded totaling 4. Confirmation of 17␣ and 17-Trenbolone is done by comparing the ratio of product ions m/z 253/199 in the samples with standards. As stated before the loss of water is not preferable, however, this loss must be used due to the absence of other diagnostic ions. Both product ions were detected in all samples. However, it was found that in nine out of 20 samples 17␣-Trenbolone could not be confirmed. For 17Trenbolone two samples could not be confirmed. The false compliant rates were 45% for 17␣-Trenbolone and 10% for
17-Trenbolone. The deviations between the observed ratio’s and tolerance values were relative small. In all cases the ratio exceeded the upper limit. In principle, this low confirmation score is surprising. It must be noted though, that the false compliant rates are based on single determinations (single injections) for both experiments. In practice based on the small deviations between observed and reference values, laboratories will always perform, at least, duplicate analyses.
4.
Conclusion
We have proposed that accurate mass measurement of a single MS ion at better than 3 ppm be allocated two identification points for confirmation. This has been proposed by others with less stringent boundaries [10,12]. We have shown the importance of measuring ion ratios and of good chromatographic separation and demonstrated the ability to do so by showing relative ion ratios within the tolerances accepted for animal residue analysis and good separation of the diastereoisomers of Trenbolone. Confirmation analysis by the proposed accurate mass criteria works well for analysis of 17␣- and 17Trenbolone in urine samples. Based on single injections, false compliant rates are slightly better than the case of LC/QqQ-MS. In 17 of 20 spiked urine samples (1 g L−1 ) the presence of 17␣Trenbolone was confirmed. In 16 of 20 samples the presence of 17-Trenbolone was confirmed. Comparison of the confirmation analysis by accurate mass with the confirmation analysis on a triple quadrupole, it was found that accurate mass gives slightly better results. Although more research is necessary to demonstrate the applicability of the proposed accurate mass criteria, these results show that the use of accurate mass measurement is a promising tool to perform confirmation analysis. For evaluating unambiguous identification a large database was used similar to libraries used in EI GC/MS [17,18]. Given the propensity of possible compounds, there could be false non-compliant results when analyzing unknown samples. However, given the retention time and ion ratio criteria this possibility is reduced dramatically (and it could be questioned whether absolute unambiguous identification is at all possible). The proposed confirmation criteria are different from another proposal [12] in respect of the use ppm error instead of fixed mass errors. We believe that the use of a relative large fixed mass error could give rise to false non-compliant results. The suggestion [12] to include a resolving power (FWHM) criteria is a good addition and should be included in the final confirmation criteria for accurate mass measurements. However, any criteria should be practical and applicable to most laboratories or it will loose its value [17]. Combining the proposed criteria would lead to the following confirmation criteria in combination with the retention time criteria for accurate mass measurement: each measured accurate mass yields two identification points if it is within 3 ppm of the theoretical mass, the resolution (FWHM) has to equal or greater than 10,000. The true value of TOF-MS in confirmation analysis, however, remains to be established.
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references [9] [1] R.A. de Zeeuw, J. Chromatogr. B Analyt. Technol. Biomed. Life Sci. 811 (2004) 3. [2] F. Andre, K.K.G. De Wasch, H.F. De Brabander, S.R. Impens, L.A.M. Stolker, L. van Ginkel, R.W. Stephany, R. Schilt, D. Courtheyn, Y. Bonnaire, P. Furst, P. Gowik, G. Kennedy, T. Kuhn, J.-P. Moretain, M. Sauer, Trends Anal. Chem. 20 (2001) 435. [3] 2002/657/EC: Commission Decision of 12 August 2002 implementing Council Directive 96/23/EC concerning the performance of analytical methods and the interpretation of results (notified under document number C (2002) 3044), OJ L221/8 (17.8.2002). [4] R. Bethem, J. Boison, J. Gale, D. Heller, S. Lehotay, J. Loo, S. Musser, P. Price, S. Stein, J. Am. Soc. Mass Spectrom. 14 (2003) 528. [5] P. Van Eenoo, F.T. Delbeke, Chromatographia 59 (2004) S39. [6] Nederlandse staatscourant, 1983, artikel 25a, lid 4, sub b. [7] Commission Decision 87/410/EEC of 14 July 1987 laying down the methods to be used for detecting residues of substances having a hormonal action and of substances having a thyrostatic action (OJ No L 223, 11.8.1987, p. 18). [8] COMMISSION DECISION 93/256/EEC of 14 April 1993 laying down the methods to be used for detecting residues of
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substances having a hormonal or a thyrostatic action (OJ No L118, 14.5.1993). A.M. Stolker, W. Niesing, E. Hogendoorn, J.M. Versteegh, R. Fuchs, U.T. Brinkman, Anal. Bioanal. Chem. 378 (2004) 955. F. Hernandez, M. Ibanez, J.V. Sancho, O.J. Pozo, Anal. Chem. 76 (2004) 4349. E.M. Thurman, I. Ferrer, J.A. Zweigenbaum, Anal. Chem. 78 (2006) 6703. M.W.F. Nielen, M.C. van Engelen, R. Zuiderent, R. Ramaker, Anal. Chim. Acta 586 (2007) 122. A.W.T. Bristow, K.S. Webb, J. Am. Soc. Mass Spectrom. 14 (2003) 1086. K. Webb, T. Bristow, M. Sargent, B. Stein, Methodology for Accurate Mass Measurement of Small Molecules—Best Practice Guide, LGC, 2004. M.H. Blokland, P.W. Zoontjes, S.S. Sterk, J. Zweigenbaum, L.A. van Ginkel, Poster on confirmatory analysis of Trenbolone using LC/TOF-MS, in: Second International Symposium on Recent Advances in Food Analysis, Prague, Czech Republic, 2005. PubChem, National Center for Biotechnology Information, http://pubchem.ncbi.nlm.nih.gov/search/search.cgi, National Institutes of Health, 2007. J.A. Sphon, J. Assoc. Off. Anal. Chem. 61 (1978) 1247. S.E. Stein, D.N. Heller, J. Am. Soc. Mass Spectrom. 17 (2006) 823.
available at www.sciencedirect.com
journal homepage: www.elsevier.com/locate/aca
Confirmatory analysis of Trenbolone using accurate mass measurement with LC/TOF-MS M.H. Blokland a,∗ , P.W. Zoontjes a , S.S. Sterk a , R.W. Stephany a , J. Zweigenbaum b , L.A. van Ginkel a a b
National Institute of Public Health and the Environment (RIVM), Bilthoven, The Netherlands Agilent Technologies, Wilmington, DE, USA
a r t i c l e
i n f o
a b s t r a c t
Article history:
The use of accurate mass measurement as a confirmation tool is examined on a TOF-MS
Received 3 December 2007
and compared with confirmation using a triple quadrupole mass spectrometer (QqQ-MS).
Received in revised form
Confirmation of the identity of a substance using mass-spectrometric detection has been
12 April 2008
described. However, the use of accurate mass measurement for confirmatory analysis has
Accepted 14 April 2008
not been taken into account. In this study, criteria for confirmation with accurate mass are
Published on line 24 April 2008
proposed and feasibility is demonstrated. Mass accuracy better than 3 ppm of the quasimolecular ion and a fragment and their relative ratios determined with LC/TOF-MS are
Keywords:
compared to the criteria of two transition ions and their ratio of LC/QqQ-MS. The results
Accurate mass measurement
show that these criteria can be met for Trenbolone in samples of bovine urine and that single
Exact mass
MS accurate mass measurement is comparable to nominal mass MS/MS for confirmation.
Time of Flight Mass Spectrometry
The increase in popularity and availability of LC/TOF-MS instruments and the ease, of which
Triple Quadrupole mass
exact masses can be measured, make it important to formulate criteria for this type of
spectrometry
instrumentation. It is shown in this study that accurate mass measurement can be used for
Confirmation
confirmatory analysis. However, more experiments need to be conducted to demonstrate the applicability of accurate mass measurement in general for residue analysis. © 2008 Elsevier B.V. All rights reserved.
1.
Introduction
In residue analysis, mass spectrometry is widely used to identify and confirm regulated and banned substances in food products, the environment, and toxicology. To identify these substances, targeted analysis strategies are typically used. In practice, samples are typically screened using a list of compounds. After identification of a compound by the screening process, confirmation is needed. Confirmation entails the utilization of an analytical method with high selectivity, usually with different characteristics than the screening method, and with direct comparison to an authentic standard. The ultimate
goal of confirmation is unambiguous identification or, put another way, the exclusion of all other possible compounds [1]. The formulation of specific criteria and use of identification points is widely adapted for compound confirmation using chromatography combined with mass spectrometry [2]. This has been written into European Union legislation and, depending on the purpose of the analysis and legal status of the analyte, it is necessary to collect three or four identification points for confirmation [3]. These points are obtained by measurement of at least two ions (in the case of MS/MS or high-resolution MS) of which their ratios are calculated and compared with those obtained from authentic standards
∗ Corresponding author at: National Institute of Public Health and the Environment (RIVM), P.O. Box 1, 3720BA Bilthoven, The Netherlands. Tel.: +31 302743966. E-mail address: [email protected] (M.H. Blokland). 0003-2670/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.aca.2008.04.040
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under the same analytical conditions. When these ratios are within predefined limits and the retention time of these ions matches the analytical standard, the required points are obtained, thus providing confirmation [4]. In view of the low concentrations in real samples at which confirmation of, e.g. growth promoting agents is required (for example biological materials obtained from cattle), the criteria for confirmation analysis using mass spectrometry are most important. The MS-identification criteria (identification points or IP) are described for different mass analyzers and depend on the method of fragmentation (tandem MS vs. single MS) and the resolution at which the ions are detected. The criteria specify that a mass spectrometer must be used in combination with a separation technique such as gas chromatography (GC) or liquid chromatography (LC). For LC/MS1 these criteria limit laboratories to certain types of instruments and because of the limitation of these instruments, it is virtually impossible to determine whether there are other than the targeted compounds present. To overcome this problem and to perform non-targeted analyses (compounds of interest but not on the screening list), alternatively TOF instruments can be used. However, TOF instruments are, according to current guidelines, treated equally as single quadrupole instruments due to their limited resolution. In practice, this means that after a non-compliant finding the samples have to be re-analysed on a tandem mass spectrometer. Resolution and mass accuracy are the two critical factors when different type of MS principles are compared and evaluated. Resolution is the degree of separation between two adjacent ions in the mass spectrum that can be distinguished at a given mass. A typical TOF mass analyzer can resolve mass differences of a few hundredths of a mass unit to a few tenths of a mass unit (e.g. 0.025–0.25 u). Mass accuracy is the difference between the theoretical (calculated) mass of a compound and the mass measured by the mass spectrometer. Mass accuracy/error can be expressed in either parts-permillion (ppm) or milli-mass units (mmu). A typical TOF can determine the accurate mass with a error of 3 ppm or better. The use of accurate mass measurement is not taken into account in current confirmation guidelines [3] and more importantly the degree of mass accuracy needed for confirmation is not specified. The closest criterion for accurate mass measurement is the high-resolution criterion defined as: In high-resolution mass spectrometry (HRMS), the resolution shall typically be greater than 10,000 for the entire mass range at 10% valley. It may be assumed that in performing highresolution mass spectrometry the MS is properly tuned and calibrated, and is capable to analyze samples within a few ppm of the theoretical exact mass. However, because a criterion for mass accuracy is not defined in the Commission Decision 2002/657/EC or other guidelines [5] it cannot be assumed that a measurement is accurate to a sufficient level no matter how great the mass resolution. Moreover, when
1
Because of the nature of fragmentation in LC/MS (low energy collision induced dissociation) for a significant number of compounds, it is impossible to obtain the four ions required with single MS and/or meet the sensitivity requirement.
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making high-resolution measurements in selected ion monitoring (SIM), an abundant co-eluting compound with a mass close to the recorded SIM mass, can result in an erroneous signal at the measured SIM mass. Criteria for accurate mass measurements were described in 1983 [6] and were included in Commission Decision 87/410/EEC [7]. These early criteria were based on mass measurements with accuracy expressed in milli Dalton error calculated from the exact mass. Unfortunately, these criteria “disappeared” in 1993 from Commission Decision 93/256/EC [8] and were not included in 2002/657/EC [3]. The use of accurate mass measurement for confirmatory analysis, however, was explored and used [9]. It was stated that mass errors below 2 mDa were generally accepted as accurate mass measurements [10]. In a recent report, accurate mass measurement was described as a valuable tool for confirmation and indicated the specificity gains from 5 ppm error to 2 ppm [11]. However, no recommendation on what would be an acceptable tolerance was given. In a recent paper [12] criteria were proposed for confirmation analysis using accurate mass measurements: for TOF instruments (mass resolving power (FWHM) ≥10,000) 1.5 point/ion or product ion with at least one ratio. As mass accuracy criterion ≤5 mDa was proposed. However, when mDa error is expressed in the commonly used ppm error [13,14] an accurate mass measurement with an error of 5 mDa for small molecules, for example 300 g mol−1 , translates to a mass accuracy of 16.6 ppm, and at mass 1000 g mol−1 it is 5 ppm. Modern instruments can do better than 3 ppm for masses below 1000 g mol−1 . For small molecules the proposed criteria of 5 mDa will generate falsely identified compounds. The use of a more strict criterion in ppm error for molecules with a mass less than 1000 gains much more selectivity than the use of a mDa error and, as we will show, may be more important for confirmation than resolving power. An exploration of the use of accurate mass measurement to gain identification points is important. The measurement of only one exact mass will not be sufficient to confirm the identity. With a typical mass accuracy of 3 ppm, several elemental compositions usually remain possible. Moreover, the measurement of a single exact mass gives no information about the structure of a compound. Therefore, measuring a fragment ion within 3 ppm of the exact mass along with the quasi-molecular ion is necessary. This gives structural information about the compound measured and correlates the proposed molecular formula of the two ions (the fragment’s ion formula must be a subset of the quasi-molecular ion formula). We and others propose that this combined with the ion-ratio criteria as already defined in CD 2002/657/EC and matching chromatographic retention time would constitute confirmation [12,15]. This work is novel in that it reports the use of a single TOFMS with the capability of routinely measuring highly accurate masses for confirmation using two ions, the quasi-molecular ion and a resulting fragment generated by in-source collision induced dissociation. This is the first report in which the ion ratios obtained from these measurements are compared with those obtained with LC/QqQ-MS. In addition, good chromatographic separation and matching retention times with that of authentic standards remains an important criterion for confirmation.
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This study investigates the use of accurate mass measurements with an LC/TOF-MS, using the above described criteria for confirmation of the identity of a compound and compares the results with those obtained by QqQ-MS/MS measurements. The relationship between mass accuracy, ion ratios, and retention-times is described and evaluated with respect to confirmation and its ultimate goal of unambiguous identification.
2.
Experimental
2.1.
Materials
All solvents used were of analytical grade or better. Deionized water was obtained from a Milli-Q purification system. The compounds 17␣- and -Trenbolone and deuterated 17Trenbolone-D3 were obtained from the “Bank of Reference Standards” (RIVM, the Netherlands). Immunoaffinity columns (IAC) were obtained from Randox (Ireland).
2.2.
Hydrolysis of urine
A test portion of 5.0 mL bovine urine was pipetted into a 50mL plastic centrifuge tube; internal standard, 50 L of 0.1 ng 17-Trenbolone-D3 L−1 , was added and mixed. The pH was adjusted to 5.2 with 2 mL acetate–buffer (2 mol L−1 ). To deconjugate glucuronide and sulfate conjugates, 75 L suc d’Helix Pomatia was added and the tube was vortexed. The mixture was incubated for 3 h at 37 ◦ C.
2.3.
Liquid/liquid extraction (LLE)
LLE was performed with 10 mL of hexane/butanol (80/20, v/v%). After centrifugation (2 min at 3000 × g), the top layer was transferred to a glass tube and evaporated to dryness under a stream of nitrogen at 55 ◦ C. The dry residue was dissolved in 250 L of ethanol after which 5 mL of water was added.
2.4.
Immunoaffinity chromatography (IAC)
The IAC-Trenbolone column was conditioned by washing twice with 5 mL of wash-buffer (provided by Randox). The total sample extract was applied to the IAC-column. After sample application the column was washed twice with 5 mL of buffer and with 5 mL of water, and eluted with 5 mL of 70/30 (v/v) ethanol/water. The eluate was evaporated to dryness under a stream of nitrogen at 55 ◦ C. The sample was redissolved in 500 L of ethanol and transferred to a 2 mL vial. The ethanol was evaporated to dryness at 50 ◦ C and 100 L acetonitrile/water (40/60, v/v) was added. The vial was vortexed for 30 s. The mixture was transferred into an insert and the vial was capped, ready for LC/MS analysis.
2.5.
LC/TOF-MS
LC/TOF-MS measurements were performed on an Agilent 6210 LC/TOF-MS coupled via an APCI interface to an Agilent LC1200 SL binary system with column oven. The LC column used for the TOF experiments was a Zorbax Rapid Resolution C18
(2.1 mm × 50 mm, d.f. = 1.8 m). Mobile phase A consisted of 10/90 (v/v) acetonitrile/water, mobile phase B of 90/10 (v/v) acetonitrile/water. The gradient used was linear, started at 40% A and progressed to 90% B in 10 min. After 10 min the mobile phase was kept for 2 min at 90% B. The column oven was set to 80 ◦ C and the mobile phase flow was set at 0.5 mL min−1 . The injection volume was 40 L. The following parameters were used for the LC/TOF-MS measurement. The gas temperature was 325 ◦ C, nitrogen drying gas was set at 10 L min−1 and the nebulizer pressure was 50 psi. The dielectric capillary exit was 250 V (fragmentor setting) and the skimmer 60 V. The octapole DC1 was set to 35.5 V and octapole RF to 250 V. The instrument was tuned and calibrated with the standard calibration solution provided by the manufacture. During analyses, an internal mass reference solution was added via a T-junction. This reference solution contained two compounds, purine and HP-921, with protonated masses of 121.0509 and 922.0098 respectively, which were used for continuous mass axis calibration. Instrument resolution (FWHM) was approximately 10,000 for mass 922.0098. Both the 17␣- and -Trenbolone were “in-source” fragmented in the high-pressure region between the dielectric capillary exit and the skimmer. The measured ions of 17␣/Trenbolone were the protonated molecule m/z 271.16926 amu and the loss of water at m/z 253.15869.
2.6.
LC/MSMS
LC/QqQ-MS measurements were performed on a Waters Micromass Ultima coupled via an ESI interface to a Waters 2695 LC system with column oven. The LC column used for the MSMS experiments was an Alltech Alltima C18 (2.1 mm × 150 mm, d.f. = 5 M). Mobile phase A consisted of 10/90 (v/v) methanol/water, mobile phase B of 90/10 (v/v) methanol/water. The gradient used was linear, started at 50% A and progressed to 80% B in 10 min. After 11 min the mobile phase was kept for 4 min at 100% B and finally returned in 0.1 min to its initial conditions. Mobile phase flow was set at 0.3 mL min−1 , the column oven to 40 ◦ C. The following parameters were used for the LC/MSMS measurement. The electrospray capillary was at 3.2 kV, the cone voltage was 50 V and the collision energy was set at 16.0 eV, argon was used as collision gas at 2.6 × 10−3 mbar. The ion source temperature was 110 ◦ C and the desolvation temperature was 350 ◦ C. Nitrogen was used as nebulizer gas, cone gas flow 113 L h−1 and desolvation gas flow 637 L h−1 . Data were acquired in the multiple reaction monitoring (MRM) mode. The precursor ion for 17␣/-Trenbolone m/z 271, two product ions m/z 199 and m/z 253, the precursor ion for 17-Trenbolone-D3 m/z 274 and one product ion m/z 256 were monitored. The dwell time was set at 100 ms for each.
2.7.
Experiments
2.7.1.
LC/TOF-MS
The exact masses of 17␣/-Trenbolone and 17-TrenboloneD3 were measured at three different levels close to the detection limit of the instrument (1–0.5–0.1 ng on column). To test the capabilities of the LC/TOF-MS for confirmation in samples of bovine urine, the accurate masses of the selected
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ions were determined in 20 different spiked samples at 1 and at 5 g L−1 respectively. After sample preparation a concentration of 1 g L−1 corresponds to the recommended value for screening and confirmation previous know as the minimum required performance limit (MRPL) for this analyte matrix combination. The accurate masses were determined by background subtraction of the signal in front of the peak. The ratios were calculated according CD 2002/657/EC.
2.7.2.
LC/MSMS
The same 20 samples of bovine urine, spiked at 1 g L−1 of 17␣and -Trenbolone and 17-Trenbolone-D3 were analysed with LC/MSMS. For confirmation, the ratios of the two product ions were calculated according to CD 2002/657/EC.
3.
Results and discussion
Using the IP system that has been implemented for confirmation of banned substances in animal and animal products is an appropriate reference for proposing accurate mass criteria. The criteria should include a tolerance for mass at better than 3 ppm; a tolerance for ion ratios, and thus at least two ions to be measured; and a requirement and tolerance for matching chromatographic retention time with a reference compound. An understanding of the need for a strict mass tolerance can be better obtained by taking the protonated exact mass of Trenbolone (C18 H23 O2 ) at m/z 271.1693. Determination of the molecular formula at this mass within 3 to 15 ppm for only the elements C,H,O will only yield C18 H22 O2 . However, this does not eliminate the possibility of a false non-compliant. If the common elements C,H,O,N, and S were included the possibilities increase to eight different molecular formulas at 15 ppm as shown in Table 1. At 3 ppm error the possible formulas are reduced to three. Although the fragment ion from water loss is not considered a unique ion, it is the major fragment of Trenbolone and indicative of a structural characteristic of the molecule. If the same criterion is applied to this fragment ion of Trenbolone, six formulas are possible as shown in Table 2. Of the six formulas, five correlate to formulas of the parent ion, m/z 271.1692, at a tolerance of 15 ppm mass error. At 3 ppm only two formulas are subsets of the parent compound and one of these two, C3 H17 N12 O2 , does not likely represent an actual chemical. Since, this formula is searched on the extensive
Table 1 – Possible formulas for Trenbolone ion (M+H)+ with tolerance to 15 ppm and elements including C,H,N,O,S Formula C18 H23 O2 C3 H19 N12 O3 C11 H23 N6 S C14 H19 N6 C7 H23 N6 O5 C6 H23 N8 O2 S C15 H27 O2 S C13 H23 N2 O4
m/z 271.1692 271.1697 271.1699 271.1665 271.1724 271.1659 271.1726 271.1652
Error (ppm) 0.0 −1.8 −2.5 9.9 −11.7 12.3 −12.4 14.8
Table 2 – Possible formulas for Trenbolone fragment ion (M+H-H2 O)+ with tolerance to 15 ppm and elements including C,H,N,O,S Formula C18 H21 O C3 H17 N12 O2 C10 H25 N2 O3 S C7 H21 N6 O4 C6 H21 N8 OS C15 H25 OS
m/z 253.1586 253.1591 253.158 253.1618 253.1553 253.162
Error (ppm) 0.0 −2.0 2.6 −12.6 13.2 −13.3
Internet accessible database, PubChem [16], no chemicals are found. This leaves, at 3 ppm error, only the correct formula for Trenbolone and demonstrates that the mass tolerance criteria needs to be restricted to this level to obtain the selectivity needed for confirmation. Having the correct molecular formula does not constitute confirmation in the sense of unambiguous identification. If the formula of the molecule, C18 H22 O2 , (obtained with an accuracy of 3 ppm for the quasi-molecular ion and a fragment) was searched in the same database, PubChem, 487 possible compounds are obtained. However, considering that phenolic OH on a molecule will not yield a water loss fragment and that typically small alkyl OH will readily loose the alcohol and not water, we can reduce the number of possible compounds from 487 to 59 containing an OH that would readily loose water. Of those 59 in the database, three are listed as Trenbolone, 17␣-Trenbolone, and 17-Trenbolone leaving 56 other compounds that could generate a false non-compliant. This approach towards showing specificity is not unique as Sphone demonstrated that the 3 ion criterion with single MS MIM was effective in searching a large library [17]. When Sphone reported his results, the library he used (MSSS) contained over 30,000 compounds. The database used here for searching unique molecular formulas contains over 10,000,000 compounds. This search shows how many possible compounds could interfere and highlights the importance of good chromatographic separation and the need to measure relative ion ratios to attain an unambiguous identification for confirmation. A similar search of mono-isotopic masses from 269.8–270.3 (nominal mass measured with triple quadrupole instruments) yields 20,878 compounds in this database. Because LC/MS/MS has become the standard for confirmation in residue analysis it becomes evident that the need for good chromatographic separation and measurement of ion ratios is just as important using this technology. Also with nominal mass triple quadrupole instruments set to unit resolution, all isotopic information is lost allowing inclusion of elements such as the halogens as possible interferences. The first experiment examined whether the identity of 17␣/-Trenbolone could be confirmed based on the proposed accurate mass criterion for LC/TOF-MS. For this purpose, low concentrations of the compounds in pure solvent were analyzed near the expected limit of detection of the analytical method. For the two ions selected, the accurate masses were measured (criterion ≤3 ppm deviation). To determine the number of identification points acquired, the same allocation as prescribed for high-resolution mass spectrometry was followed [3]. Each measured accurate mass yielded 2 points
3.3 19.2 5 0 1.0 14.6 10 4 0.5 10 8 17-Trenbolone 271.1693 253.1514
14.2
7.8 18.2 5 2 1.3 19.2 10 6 0.5 19.5 10 10 17␣-Trenbolone 271.1693 253.1587
Average ratio (%) Number < 3 ppm Number < 3 ppm Average ratio (%)
Uncertainty (S.D.%) Number < 3 ppm
Average ratio (%)
Uncertainty (S.D.%)
0.1 ng (n = 10) 0.5 ng (n = 10) 1.0 ng (n = 10)
if it was within 3 ppm of the theoretical mass. Using these standards a ratio criterion must be established. Thus, the repeatability of the ratio is very important. It is on the basis of the average value of the ratio for the standards that unknown signals are evaluated. Therefore, the uncertainty of the average value as determined for the standard substance must be small in comparison with the tolerance interval allowed. In Table 3 an overview of the results for the standards is shown. From Table 3 it is observed that at 1 ng on-column it is possible to measure the relative intensity with very good reproducibly. For 17␣-Trenbolone, the most dominant metabolite of Trenbolone, when analyzing samples of urine, all measured masses were within 3 ppm of the theoretical value. In two samples the error for m/z 253 for 17-Trenbolone was larger then 3 ppm. This gives in total 18 successful determinations within 20 tests. At 0.5 ng on-column, however, the mass accuracy was not within 3 ppm for 10 individual measurements out of 40 determinations and at 0.1 ng on-column 28 determinations failed. This failing of confirmation in these cases is due to the low signal intensity of the ions. Therefore, it is concluded that 1 ng on-column is the lower limit for confirmation using the criterion of ≤3 ppm mass measurement error. Furthermore, Table 3 shows that the repeatability of the ratios is acceptable for the standards at 1.0 and 0.5 ng oncolumn, with only small differences between these amounts tested. The repeatability for the standard at 0.1 ng is not as good, and again this can be attributed to the low signal for this concentration of the standard. From the experiment with standards, it can be concluded that reliable confirmation is possible when approximately 1 ng 17␣- or 17-Trenbolone is injected on-column. Based on a minimum recommended value of 1 g L−1 for both compounds and a sample portion of 5 mL, this is sufficient. However, other factors such as matrix interferences could reduce the sensitivity in real samples. For testing of the confirmation capabilities of the LC/TOF-MS, 20 different samples of bovine urine were spiked at 1 g L−1 . In Fig. 1 a typical chromatogram and spectrum are shown for a sample of urine spiked at 1 g L−1 . The loss of water (m/z 271 → 253) is in general not considered a specific loss, however, if the accurate mass of this fragment ion is within the subset of the fragment’s of the quasi-molecular ion, it can be considered as diagnostic. Of course if there are other fragment ions available within the subset of the quasimolecular ion these also should be considered. As can be seen from the chromatogram and spectrum no matrix interferences are visible. Based on these data, it should be possible to confirm the identity at mass concentrations of 1 g L−1 as compared to the standard at 1 ng on-column. The results of the accurate mass measurements and their ion ratios of the spiked urine samples are given in Table 4. For 17␣-Trenbolone, all measured masses for m/z 271 are within 3 ppm of the theoretical values and therefore fulfill the mass accuracy criterion. However, 3 measured masses for m/z 253 deviate by more then 3 ppm and therefore failed the mass accuracy criterion. The ratios of 271/253 are all within the limits of the reference value as given in the 2002/657/EC. Therefore, in total, the identity of 17␣-Trenbolone was confirmed in 17 out of 20 samples. The false compliant rate (<4 IP), therefore, is 15%. For 17-Trenbolone one ion at m/z of 271 and
Uncertainty (S.D.%)
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Table 3 – Results of the accurate mass measurement of 10 injections of the test compounds in pure solvent at three different levels (1, 0.5, 0.1 ng on-column)
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Fig. 1 – (A) LC/TOF-MS chromatogram of 17␣/-Trenbolone. (B) Mass spectrum of 17-Trenbolone, arrows point to the m/z used for confirmation analysis.
three at m/z 253 did not fulfill the accurate mass measurement criterion. Nevertheless, all relative ratios for 17-Trenbolone were within the limits given in CD 2002/657/EC. Therefore, in 20% of these samples a false compliant result (<4 IP) was obtained.
Thus, for confirmation with LC/TOF-MS in 20 samples containing two compounds each, seven instances failed. In those seven cases, the mass deviation was between 3 and 5 ppm for 5 measurements. In the other two cases, the deviation was higher, with a maximum measured deviation of 7.74. This
Table 4 – Determination of the exact mass and ion ratio in 1 g L−1 spiked urine samples 17␣-Trenbolonea (271.169, <3 ppm) −0.81 −0.74 −2.29 −1.99 0.41 0.41 −0.22 −0.44 −0.33 −1.66 −0.44 −0.37 0.55 0.18 0.00 0.96 0.63 0.81 1.81 1.14 a b
17-Trenboloneb (253.158, <3 ppm) 0.16 1.66 −1.22 −2.09 2.61 2.13 −0.71 0.40 1.82 0.95 1.15 2.05 3.87 3.28 2.41 2.77 2.92 2.80 4.15 2.61
Ratio 271/253 = 18.0 18.6 16.0 18.2 16.9 18.3 17.6 18.7 18.8 17.2 19.3 16.8 17.1 17.6 18.1 16.0 18.2 17.4 20.2 18.0 18.9
IP
271.169, <3 ppm ()
253.158, <3 ppm ()
4 4 4 4 4 4 4 4 4 4 4 4 2 2 4 4 4 4 2 4
0.32 −0.33 −5.31 −1.92 0.48 −2.77 −1.00 −2.51 −0.81 1.33 −1.99 −1.88 0.07 0.89 0.77 0.77 −0.92 −0.52 1.51 1.18
−0.48 4.70 −2.84 2.69 2.69 −0.36 2.49 0.79 2.45 −2.53 1.15 0.16 2.29 2.92 0.83 3.00 3.36 1.30 7.74 2.13
Ratio 271/253 = 12.9 12.3 12.5 12.9 14.5 12.9 13.0 13.2 12.7 13.4 13.2 12.7 12.2 12.6 12.9 12.3 12.9 13.1 13.1 13.3 13.4
IP 4 4 2 4 4 4 4 4 4 4 4 4 4 4 4 4 2 4 2 4
17␣-Trenbolone: lower limit 12.6; upper limit 23.4. 17-Trenbolone: lower limit 9.1; upper limit 16.8. Lower and upper limits were calculated according Commission Decision 2002/657 (±30% for ions with intensity between 10 and 20%).
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Table 5 – Results of the ratio for two product ions obtained for samples of spiked bovine urine (1 g L−1 ) on a LC/QqQ-MS 17␣-Trenbolonea 253/199 = 19.0 22.4 21.4 21.4 22.0 24.6 26.6 23.9 23.3 26.4 24.4 24.5 24.7 25.6 25.1 30.4 27.4 27.3 28.3 25.7 21.0 a b
Ratio
17-Trenboloneb Ratio 253/199 = 66.2. 67.5 69.6 75.4 65.6 57.3 63.2 75.6 71.4 71.3 74.5 56.4 54.3 61.6 82.1 67.8 61.7 59.2 83.6 62.8 79.0
17␣-Trenbolone: lower limit 13.3; upper limit 24.7. 17-Trenbolone: lower limit 52.7; upper limit 79.0. . Lower and upper limits were calculated according Commission Decision 2002/657.
is considerably higher than expected based on the measurements made on standards. To check whether the failing of the 3 ppm criterion for confirmation was due to ion-suppression or matrix effects, the same set of urine samples was spiked at 5 g L−1 . In all of these samples the accurate mass criterion was met, and the ratio was well within acceptable tolerance as described in 2002/657/EC (data not shown). To compare the effectiveness of confirmation with a widely accepted technique, the same experiment was repeated on a LC/QqQ-MS system. The results are summarized in Table 5. The criteria for MS/MS specify two product ions are to be measured at the correct chromatographic retention time. The presence of a fragment ion is assessed based on the S/N at the relevant retention time. As a rule, a S/N value of 6–10 is necessary for reliable quantification of the area. This quantification is necessary for determination of the ratio of the two product ions. The two fragment ions each only yield 1.5 IP each. However, the precursor ion is assumed because the first mass filter is set to only let that m/z pass through and thus 1 additional IP is awarded totaling 4. Confirmation of 17␣ and 17-Trenbolone is done by comparing the ratio of product ions m/z 253/199 in the samples with standards. As stated before the loss of water is not preferable, however, this loss must be used due to the absence of other diagnostic ions. Both product ions were detected in all samples. However, it was found that in nine out of 20 samples 17␣-Trenbolone could not be confirmed. For 17Trenbolone two samples could not be confirmed. The false compliant rates were 45% for 17␣-Trenbolone and 10% for
17-Trenbolone. The deviations between the observed ratio’s and tolerance values were relative small. In all cases the ratio exceeded the upper limit. In principle, this low confirmation score is surprising. It must be noted though, that the false compliant rates are based on single determinations (single injections) for both experiments. In practice based on the small deviations between observed and reference values, laboratories will always perform, at least, duplicate analyses.
4.
Conclusion
We have proposed that accurate mass measurement of a single MS ion at better than 3 ppm be allocated two identification points for confirmation. This has been proposed by others with less stringent boundaries [10,12]. We have shown the importance of measuring ion ratios and of good chromatographic separation and demonstrated the ability to do so by showing relative ion ratios within the tolerances accepted for animal residue analysis and good separation of the diastereoisomers of Trenbolone. Confirmation analysis by the proposed accurate mass criteria works well for analysis of 17␣- and 17Trenbolone in urine samples. Based on single injections, false compliant rates are slightly better than the case of LC/QqQ-MS. In 17 of 20 spiked urine samples (1 g L−1 ) the presence of 17␣Trenbolone was confirmed. In 16 of 20 samples the presence of 17-Trenbolone was confirmed. Comparison of the confirmation analysis by accurate mass with the confirmation analysis on a triple quadrupole, it was found that accurate mass gives slightly better results. Although more research is necessary to demonstrate the applicability of the proposed accurate mass criteria, these results show that the use of accurate mass measurement is a promising tool to perform confirmation analysis. For evaluating unambiguous identification a large database was used similar to libraries used in EI GC/MS [17,18]. Given the propensity of possible compounds, there could be false non-compliant results when analyzing unknown samples. However, given the retention time and ion ratio criteria this possibility is reduced dramatically (and it could be questioned whether absolute unambiguous identification is at all possible). The proposed confirmation criteria are different from another proposal [12] in respect of the use ppm error instead of fixed mass errors. We believe that the use of a relative large fixed mass error could give rise to false non-compliant results. The suggestion [12] to include a resolving power (FWHM) criteria is a good addition and should be included in the final confirmation criteria for accurate mass measurements. However, any criteria should be practical and applicable to most laboratories or it will loose its value [17]. Combining the proposed criteria would lead to the following confirmation criteria in combination with the retention time criteria for accurate mass measurement: each measured accurate mass yields two identification points if it is within 3 ppm of the theoretical mass, the resolution (FWHM) has to equal or greater than 10,000. The true value of TOF-MS in confirmation analysis, however, remains to be established.
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