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Fast Screening of Recalled Tylenol for Tribromoanisole and Related Adulterants Using QuEChERS and GC-TOFMS

By Jason Thomas, Innovations Chemist, and Jack Cochran
  • Rapid sample preparation with QuEChERS improves turnaround time for emergency response analysis situations.
  • Prepackaged QuEChERS extraction salts and snap-and-shoot standards reduce human error and save time.
  • Rugged, inert, thermally stable Rxi-5Sil MS column extends applicability to acids, bases, and higher molecular weight adulterants.

Introduction

The recent recall of Tylenol pain reliever and other related products highlights the need for simple, quick sample preparation and a comprehensive analytical method for adulterants in consumer products. The rush to examine a multitude of samples in a short period of time is a common scenario for potential recalls, especially when a contaminant is found in a given product and rapid determinations need to be made to assess how widespread the problem may be.

The QuEChERS (Quick, Easy, Cheap, Effective, Rugged, and Safe) sample preparation approach, originally developed to prepare fruit and vegetable samples for pesticide residue analysis, is being adopted for other applications and may be useful when rapid screening methods are required. QuEChERS employs a simple solvent shake and centrifugation step, with an optional dispersive solid phase extraction (dSPE) cleanup. In addition to being quick and easy, the use of acetonitrile in QuEChERS allows compounds containing a wide variety of chemical functionalities to be extracted, which is very important when trying to isolate an unknown adulterant. The resulting extract is appropriate for both GC/MS and LC/MS work.

The utility of the QuEChERS method is illustrated here using the aforementioned Tylenol example, showing the applicability to this problem and, by extension, to others like it. This particular recall was due to the presence of 2,4,6-tribromoanisole (TBA) causing a musty smell in the product and, in some cases, nausea in the consumer [1]. TBA is a known breakdown product of 2,4,6-tribromophenol (TBP), which is a fumigant used on shipping pallets; TBA production occurs through a process actuated by a fungus, Paecilomyces variotii [2]. TBA is a common and undesirable odorant in the winemaking industry where it and similar compounds (e.g. trichloroanisole) create a situation known as cork taint [3].

This work demonstrates the potential applicability of QuEChERS sample preparation and GC-TOFMS analysis to screening methods for anisole contaminants. Advantages of methods developed based on QuEChERS and GC-TOFMS may include rapid sample screening and definitive identifications in the presence of significant amounts of matrix.

Procedure

TBA, TBP, 2,3,4,5-tetrachloroanisole, and pentachloroanisole were spiked into ground up Tylenol caplets at two different concentrations and extracted using QuEChERS. Several cleanup procedures were performed for comparison and GC analysis was conducted using a sensitive, full mass-range time-of-flight MS.

Sample Wetting and Fortification

A bottle of recalled Tylenol Extra Strength caplets was used for this work, although no odor of TBA was detected. Multiple caplets were ground to a fine powder using a Bamix Mono Hand Mixer with dry grinder attachment. 1.2 g of powder, equivalent to 2 caplets (500 mg acetaminophen each) was wetted with 9 mL organic-free water for each sample for extraction. After shaking to mix well, wetted powders were fortified as follows; note that spike levels are expressed relative to approximated amount of active ingredient, not formulated product.

  • Unspiked Tylenol
    100 µL of QuEChERS Internal Standard Mix for GC/MS Analysis (cat.# 33267)
    containing PCBs 18, 28, and 52 (50 µg/mL each); triphenylphosphate (20 µg/mL); tris-(1,3-dichloroisopropyl)phosphate (50 µg/mL); and triphenylmethane (10 µg/mL).
  • ~1,000 ng/g spiked Tylenol (2 samples)
    5 µL of Custom Anisoles Standard #1 (cat.# 564667) containing 2,4,6-tribromoanisole, 2,3,4,5-tetrachloroanisole, and pentachloroanisole at 200 µg/mL each in methanol. 5 µL of Acid Surrogate Mix (cat.# 31025) containing 2,4,6-tribromophenol, 2-fluorophenol, and phenol-d6, diluted to 200 µg/mL in methanol. 100 µL of QuEChERS Internal Standard Mix for GC/MS Analysis.
  • ~100 ng/g spiked Tylenol
    5 µL of Custom Anisoles Standard #1; 5 µL of Acid Surrogate Mix diluted to 20 µg/mL; 100 µL of QuEChERS Internal Standard Mix for GC/MS Analysis.

After fortification, each sample was allowed to soak for 1 hour prior to QuEChERS extraction. Originally the QuEChERS method was developed for high aqueous content fruits and vegetables. For a dry powder, a reduced amount of material, and wetting, increases extraction efficiency.

QuEChERS Extraction

The EN 15662 QuEChERS method was used for sample extraction [4]. 10 mL of acetonitrile was added to a wet sample. After a 1 minute shake, Q-sep Q110 buffering extraction salts (cat.# 26235, 4 g MgSO4, 1 g NaCl, 1 g trisodium citrate dihydrate, 0.5 g disodium hydrogen citrate sesquihydrate) were added. Following another 1 minute shake, the sample was centrifuged for 5 minutes at 3,000 U/min. with a Q-sep 3000 centrifuge (cat.# 26230).

Extract Cleanup

Four dispersive solid phase extraction methods (dSPE) were compared. For each, 1 mL portions of QuEChERS extracts were added to tubes containing drying agent and different sorbents such as primary secondary amine (PSA), C18, and graphitized carbon black (GCB) as shown below. The tubes were shaken for 2 minutes and then centrifuged for 5 minutes in the Q-sep 3000 centrifuge. The resulting final extracts were then analyzed with GC-TOFMS.

  • Q210 (cat.# 26215): 150 mg MgSO4, 25 mg PSA
  • Q251 (cat.# 26125): 150 mg MgSO4, 50 mg PSA, 50 mg C18
  • Q252 (cat.# 26219): 150 mg MgSO4, 50 mg PSA, 50 mg C18, 50 mg GCB
  • Custom dSPE tube: 150 mg MgSO4, 50 mg PSA, 50 mg C18, 7.5 mg GCB

GC-TOFMS

A LECO Pegasus III GC-TOFMS instrument was used and all data were processed with LECO ChromaTOF software. Gas chromatography was performed using an Rxi-5Sil MS column (30 m x 0.25 mm x 0.25 µm, cat.# 13623) with a constant flow of helium at 1.2 mL/min. (40 cm/sec. at 90°C). 1 µL fast autosampler splitless injections were made into a 4mm single gooseneck liner with wool (cat.# 22405) at 250°C. The purge valve time was 60 seconds.

The GC oven program was 90°C (1 minute), 4°C/min. to 310°C (2 minutes). Total run time was 58 minutes.

Electron ionization at 70 eV was used with a source temperature of 225°C. Data acquisition was from 45 to 550 amu at a rate of 5 spectra/sec.

Calibration and Quantification with Matrix-Matched Standards

Matrix-matched standards were prepared at 100 pg/µL and 10 pg/µL, as these are the expected final concentrations in extracts for Tylenol spikes (assuming 100% recoveries for the 1,000 and 100 ng/g spikes, respectively). Matrix-matched standards were prepared by adding standard solution to the final extract from a control sample, which had no measurable amounts of the compounds of interest. Actual recoveries were calculated after quantification from one-point calibration in ChromaTOF. The internal standard method of quantification was employed using PCB 52.

Results

The concentrations used for spikes in this case were 1,000 and 100 ng/g relative to active ingredient in the starting caplet material (estimated using labeled value). Using QuEChERS combined with GC-TOFMS, modest recoveries of all compounds were realized as can be seen in Table I. In addition, results for duplicate extracts and cleanups for 1,000 ng/g spikes, using either Q210 dSPE tubes or the custom dSPE tubes, were relatively close for each analyte. Although the spiked concentrations are higher than the odor threshold expected for an end product such as Tylenol (TBA’s odor threshold is extremely low, 0.008-0.03 ppt in water and 2-6 ppt in wine [5]), the QuEChERS approach with GC-TOFMS provides a useful technique for screening of contamination at potential levels of health concern, moderate adulteration, and for analyzing source materials such as wood pallets, for contaminants. QuEChERS can produce extracts for up to 24 samples, ready for GC or LC analysis, in less than 60 minutes, a speed conducive to the pressure of responding to a consumer product adulteration issue. In addition, the multi-compound extraction capability of the QuEChERS acetonitrile solvent offers a better chance of isolating potential adulterants from any matrix.

Table 1  Percent recoveries of potential adulterants from QuEChERS extractions: comparison of various dSPE cleanup procedures. (All samples are 1,000 ng/g, unless otherwise noted.)

Compound RT
(sec)
Q210
Q251
Q252
Custom dSPE
Extract
Sample
1
Sample
2
Sample
1
Sample
1
Sample
1
Sample
2
1,000 ng/g 100 ng/g
2,4,6-Tribromoanisole1097.82
82
56
62
68
73
68
59
51
2,4,6-Tribromophenol1133.62
55
60
40
49
66
53
63
110
2,3,4,5-Tetrachloroanisole1162.22
71
63
64
64
75
63
67
70
Pentachloroanisole1256.82
70
67
64
70
71
61
65
60
PCB 52 (IS)1611.02               
Q210 = 150 mg MgSO4, 25 mg PSA
Q251 = 150 mg MgSO4, 50 mg PSA, 50 mg C18
Q252 = 150 mg MgSO4, 50 mg PSA, 50 mg C18, 50 mg GCB
Custom = 150 mg MgSO4, 50 mg PSA, 50 mg C18, 7.5 mg GCB
Extract = extraction only, no clean up step was performed

The original QuEChERS approach for fruits and vegetables was developed with a novel dSPE cleanup procedure where an extract is shaken with loose sorbent material (e.g. primary secondary amine, C18, graphitized carbon black) to remove matrix coextractives like fatty acids, lipids, and pigments, that might interfere with targeted residues during instrumental analysis. Although we tried dSPE here, it is less appropriate in this application for two reasons: (1) In a true unknown adulterant situation, sorbents, especially PSA and GCB, might actually remove the adulterant from the extract, in addition to matrix interferences, leaving the adulterant undetected during instrumental analysis. (2) The gross amount of acetaminophen in the extract greatly exceeds the capacity of the dSPE sorbent, which is typically on the order of the 25-50 mg per mL extract.

Due to the overwhelming concentration of acetaminophen in the caplet powder extracts, dSPE cleanup was largely ineffective (Figure 1), but as the acetaminophen was volatile enough to chromatograph, it was not critical to remove it to prevent deposition in the injector and column. Elimination of the dSPE step did not noticeably improve, or degrade, the recovery results for TBA and TBP, or other components (Table I).

Figure 1  Chromatograms of QuEChERS extracts of Tylenol with different dSPE cleanups.

QuEChERS Extracts of Tylenol with Different dSPE Cleanups on Rxi-5Sil MS
GC_GN1151
ColumnRxi-5Sil MS, 30 m, 0.25 mm ID, 0.25 µm (cat.# 13623)
SampleQuEChERS extracts of Tylenol spiked with TBA and anisoles
Diluent:Acetonitrile
Injection
Inj. Vol.:1 µL splitless (hold 1 min)
Liner:Gooseneck Splitless (4mm) w/Wool (cat.# 22405)
Inj. Temp.:250 °C
Purge Flow:40 mL/min
Oven
Oven Temp.:90 °C (hold 1 min) to 310 °C at 4 °C/min (hold 2 min)
Carrier GasHe, constant flow
Flow Rate:1.2 mL/min
Linear Velocity:40 cm/sec
DetectorTOFMS
Transfer Line Temp.:290 °C
Analyzer Type:TOF
Source Temp.:225 °C
Electron Energy:70 eV
Mass Defect:-20 mu/100 u
Solvent Delay Time:2 min
Tune Type:PFTBA
Ionization Mode:EI
Acquisition Range:45 to 550 amu
Spectral Acquisition Rate:5 spectra/sec
InstrumentLECO Pegasus 4D GCxGC-TOFMS
NotesQ210 = PSA
Q251 = PSA and C18
Q252 = PSA, C18, and GCB
Custom = PSA, C18, and less GCB
(See Application Note GNAN1263 for complete description of spiking, extraction, and dSPE treatments.)

One reason to employ dSPE, or another cleanup step, is to remove matrix interferences that can prevent detection of potential adulterants. However, we relied on automated peak find and spectral deconvolution to detect analytes of interest among the overwhelming acetaminophen response. This is particularly evident for pentachloroanisole in the 1,000 ppb spike extract, which eluted well underneath the large acetaminophen peak (Figure 2). The disparity in concentrations is so large that the 265 m/z ion was only visible by magnifying it by 50, yet ChromaTOF automatically located the peak and produced a deconvoluted spectrum that matched very well with the pentachloroanisole reference spectrum (Figure 3). Although this part of the application was a targeted analysis of TBA, TBP, and other anisoles, to help evaluate QuEChERS extract recoveries for these compounds in a difficult matrix, the peak find and spectral deconvolution algorithms employed here are very useful when looking for unknown contaminants. Pure sample mass spectra lead to better library searching and identification of components.

Figure 2  Pentachloroanisole was located under the large acetaminophen peak using an automated peak find routine. (1,000 ng/g; QuEChERS extraction only, no dSPE).

QuEChERS Extracts of Tylenol Spiked with Anisoles and TBA on Rxi-5Sil MS
GC_GN1152
ColumnRxi-5Sil MS, 30 m, 0.25 mm ID, 0.25 µm (cat.# 13623)
SampleQuEChERS extracts of Tylenol spiked with TBA and anisoles
Diluent:Acetonitrile
Injection
Inj. Vol.:1 µL splitless (hold 1 min)
Liner:Gooseneck Splitless (4mm) w/Wool (cat.# 22405)
Inj. Temp.:250 °C
Purge Flow:40 mL/min
Oven
Oven Temp.:90 °C (hold 1 min) to 310 °C at 4 °C/min (hold 2 min)
Carrier GasHe, constant flow
Flow Rate:1.2 mL/min
Linear Velocity:40 cm/sec
DetectorTOFMS
Transfer Line Temp.:290 °C
Analyzer Type:TOF
Source Temp.:225 °C
Electron Energy:70 eV
Mass Defect:-20 mu/100 u
Solvent Delay Time:2 min
Tune Type:PFTBA
Ionization Mode:EI
Acquisition Range:45 to 550 amu
Spectral Acquisition Rate:5 spectra/sec
InstrumentLECO Pegasus 4D GCxGC-TOFMS
NotesTotal ion chromatogram (pink) of QuEChERS extracts of Tylenol spiked with anisoles and TBA, and extracted ion chromatogram (blue, 265x50) of pentachloroanisole, which was located by an automated peak find routine underneath the large acetaminophen peak. No dSPE was performed on this sample; see Applications Note GNAN1263 for complete sample fortification and extraction details.

Figure 3  The caliper spectrum taken at the peak apex of pentachloroanisole is representative of the overwhelming acetaminophen peak, but TOFMS allows spectral deconvolution to produce a sample spectrum that matches well with the reference spectrum.

Mass Spectra of Pentachloroanisole QuEChERS Extracts of Tylenol Spiked with Anisoles and TBA on Rxi-5Sil MS
GC_GN1153
ColumnRxi-5Sil MS, 30 m, 0.25 mm ID, 0.25 µm (cat.# 13623)
SampleQuEChERS extracts of Tylenol spiked with TBA and anisoles
Diluent:Acetonitrile
Injection
Inj. Vol.:1 µL splitless (hold 1 min)
Liner:Gooseneck Splitless (4mm) w/Wool (cat.# 22405)
Inj. Temp.:250 °C
Purge Flow:40 mL/min
Oven
Oven Temp.:90 °C (hold 1 min) to 310 °C at 4 °C/min (hold 2 min)
Carrier GasHe, constant flow
Flow Rate:1.2 mL/min
Linear Velocity:40 cm/sec
DetectorTOFMS
Transfer Line Temp.:290 °C
Analyzer Type:TOF
Source Temp.:225 °C
Electron Energy:70 eV
Mass Defect:-20 mu/100 u
Solvent Delay Time:2 min
Tune Type:PFTBA
Ionization Mode:EI
Acquisition Range:45 to 550 amu
Spectral Acquisition Rate:5
InstrumentLECO Pegasus 4D GCxGC-TOFMS
NotesThe caliper spectrum taken at the peak apex of pentachloroanisole is representative of the overwhelming acetaminophen peak, but TOF-MS allows spectral deconvolution to produce a sample spectrum that matches well with the reference spectrum. No dSPE was performed on this sample; see Applications Note GNAN1263 for complete sample fortification and extraction details.

Conclusions

Shown here is a QuEChERS multi-compound extraction method that rapidly produces samples for GC or LC analysis in consumer product adulteration cases. QuEChERS is simple, efficient, and uses little solvent compared to other extraction methods. QuEChERS and GC with a sensitive, full mass-range TOFMS is a powerful approach to identifying potential adulterants in consumer products.

REFERENCES

[1] P. Kavilanz, CNN.Money.com (2010).
http://money.cnn.com/2010/01/15/news/companies/over_the_counter_medicine_recall/ (accessed April 19, 2010).
[2] R. Tracy, B. Skaalen, Practical Winery and Vineyard (2008)
http://www.practicalwinery.com/novdec08/page2.htm (accessed April 19, 2010).
[3] F.B. Whitfield, J.L. Hill, K.J. Shaw, J. Agric. Food Chem. 45 (1997) 889.
[4] Foods of Plant Origin—Determination of Pesticide Residues Using GC-MS and/or LC-MS/MS Following Acetonitrile Extraction/Partitioning and Clean-up by Dispersive SPE (QuEChERS-method). (EN 15662 Version 2008.).
[5] P. Chatonnet, S. Bonnet, S. Boutou, J. Agric. Food Chem. 52 (2004) 1255.

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