Dyes in Fish

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A Simple SPE Method for the Determination of Malachite Green, Crystal Violet and Other Synthetic Dyes in Seafood Coupled with LC-MS/MS Detection

UCT Part Numbers: ® CSDAU206 - CLEAN SCREEN DAU 200mg / 6mL column

October 2013

Introduction

Triphenylmethane dyes (TPMs) are synthetic dyes used for a wide range of industrial applications. However, they are also used in aquaculture production due to their anti- bacterial, anti-fungal and anti-parasitic properties. They are cheap, very effective and readily available, but due to their toxicity to humans (mutagenic & carcinogenic) the TPMs are banned in the US and EU. In both regions there is a zero tolerance policy regarding their use. As such, the respective authorities (FDA and EU Commission) have established very low detection limits for these compounds in seafood products, namely 1 µg/kg (US) and 2 µg/kg (EU). As the parent dyes are rapidly metabolized to form their leuco- metabolites that predominate and persist in fish tissue, the detection limits are expressed as the sum of parent drug and corresponding leuco-form.

Analyzing TPMs can be difficult due to their instability, which causes problems during sample preparation and instrumental analysis. The TPM dyes readily undergo oxidation- reduction reactions and are prone to UV degradation (demethylation). As such, it is important to take these issues into account when developing a method or when doing routine analysis of these compounds. To stabilize the dyes it is common to add an antioxidant prior to sample extraction and in the final sample extract prior to detection. Common antioxidants used include TMPD*, hydroxylamine and ascorbic acid. Analytical standards solutions also experience stability issues and should be prepared in an aprotic solvent (e.g. acetonitrile) as the leuco metabolites are not stable for a long period of time in H2O and MeOH (even at -20 °C) and oxidize to the non-leuco form. This can in turn affect the reliability of the analytical method by leading to poor recoveries/reproducibility and non-linear calibration curves. It is also preferable to prepare a mixed working standard solution on a regular basis.

Most reported methods for TPM analysis use lengthy sample preparation procedures or do little to no sample cleanup. Most methods include malachite green, leucomalachite green, crystal violet, leucocrystal violet and sometimes the structurally related dye brilliant green. HPLC-UV is often used for their analysis due to the compounds good UV absorbance, but this technique can only detect the parent compounds and not the leuco- metabolites. To include these, an oxidation step needs to be incorporated into the LC system using a post-column reactor to convert the leuco- form to the chromophorous parent form. Alternatively, the use of LC-MS/MS avoids this requirement and is capable of detecting all TPM dyes. It also offers the advantage of simpler sample preparation methodology and the inclusion of additional compounds besides the classical TPM dyes.

Sample preparation approaches for TPM analysis often use time consuming, complicated procedures consisting of numerous steps and requiring large volumes of solvent to evaporate. SPE using strong-cation exchange sorbent is also used and is very effective at removing matrix interferences. However, it is difficult to elute dyes from SCX sorbent as most dyes carry a permanent positive charge (see Figure 1). Most methods using SCX SPE use a high salt concentration (> 1 M) for elution. However this is not very suitable for LC-MS/MS analysis due to the lack of volatility of such high salt concentrations.

This application note uses an alternative elution solvent composed of methanol (MeOH) containing 1% triethylamine (TEA) and 0.5% formic acid (i.e. triethylammonium formate). The positively charged TEA (a quaternary amine) then acts as a counter-ion to release the dyes from the sorbent. No evaporation step is included in the method which avoids any loss that may occur with this step. The eluted extracts can be analyzed directly as TEA is LC-MS/MS compatible (due to its volatility) and has no ion-pairing effect on the dyes as they carry the same charge. Ascorbic acid is used as antioxidant because positively charged hydroxylamine (quaternary amine) will interfere with the retention on the sorbent (same mechanism as elution solvent). The simple method outlined here allows for the rapid analysis of dyes in seafood while achieving good accuracy/precision and low sensitivity.

Figure 1. Structure of the 9 dyes included in the method.

Compounds included in method Abbreviation Malachite Green MG

Leuco‐Malachite Green LMG Crystal Violet CV Leuco‐Crystal Violet LCV Nile Blue NB Azure B AB Methylene Blue MB Brilliant Green BG Victoria Blue VB

Procedure

1. Sample Extraction a) Weigh 2 ± 0.1g of sample into a 15mL polypropylene centrifuge tube. b) Add 10 mL of 1% formic acid in acetonitrile and 1mL of 1M ascorbic acid (antioxidant) to each sample. c) Shake on a SPEX® SamplePrep® GenoGrinder® (or alternative mechanical shaker) or vortex samples for 15 minutes to fully extract the dyes. d) Centrifuge the samples for 10 min at ≥3000 rcf and 4°C. e) Transfer the supernatant to a 50mL polypropylene centrifuge tube and add 20mL of McIlvaine’s buffer (0.1M, pH 3.5)**. f) Vortex the samples briefly (1 min) to mix the organic solvent with the buffer. g) Prior to the SPE step, centrifuge the samples for 5 min at ≥3000 rcf and 4°C. This makes the SPE step easier by separating out any solid particles and/or lipids in the sample.

2. Condition Cartridge a) Add 3 mL of methanol to SPE cartridge CSDAU256. b) Add 3 mL of ultrapure water. c) Add 1 mL of McIlvaine’s buffer (0.1M, pH 3.5). Note: Do not let the cartridge go dry otherwise repeat steps a) through c).

3. SPE Extraction a) Load supernatant from step 1.g). b) Adjust vacuum for flow of 1–3 mL per minute.

4. Wash Cartridge a) Add 3 mL of 0.1% formic acid and slowly draw through. b) Add 3 mL of 0.1% formic acid in MeOH and slowly draw through. c) Dry under vacuum for 1 minute to remove excess solvent. Note: A portion of the dyes will stick to the SPE frit due to the hydrocarbon backbone. The wash steps included in the method uses acidified solutions to maintain a positive charge on the dyes, which ensures they are retained on the cation-exchange sorbent.

5. Elute Cartridge a) Elute the dyes from the SPE cartridge using 3-4 mL elution solvent (1% TEA + 0.5% formic acid in MeOH). b) Vortex the samples for 2 min and transfer a 1 mL aliquot to an autosampler vial for analysis.

LC‐MS/MS Conditions: HPLC Conditions Instrumentation Thermo ScientificTM DionexTM UltimateTM 3000 LC system HPLC column Thermo ScientificTM AccucoreTM C18, 100 x 2.1 mm, 2.6 µm (p/n: 17126‐102130) Guard column AccucoreTM C18 DefenderTM, 10 x 2.1 mm, 2.6 µm (p/n: 17126‐012105) Run time 12 min (including re‐equilibration time) Column temp. 40°C Injection volume 5 µL Autosampler temp. 10°C Wash solvent MeOH:ultrapure water (1:1, v/v) Flow rate 400 µL/min Divert valve Mobile phase was sent to waste (0–3.5 & and 8–12min) to prevent ion source contamination.

Mobile Phase & Gradient: A 0.1% formic acid in ultrapure water B 0.1% formic acid in MeOH Time (min) A (%) B (%) 0.0 90 10 2.0 90 10 3.0 40 60 4.0 40 60 5.0 0 100 8.0 0 100 8.2 90 10 12.0 90 10

MS Conditions Instrumentation Thermo ScientificTM TSQ VantageTM tandem mass spectrometer Ionization mode ESI+ Spray voltage 4000 V Vaporizer temperature 350°C Capillary temperature 300°C Sheath gas pressure 60 arbitrary units Auxiliary gas pressure 5 arbitrary units Ion sweep gas 0 arbitrary units Declustering potential 0 V Q1 and Q3 peak width 0.2 and 0.7 Da Collision gas Argon Collision gas pressure 2.0 mTorr Acquisition method EZ method (SRM) Cycle time 0.6 sec Software for data processing TraceFinderTM version 3.0 Weighting factor applied to calibration curves 1/X

SRM Transitions

Analyte tR (min) Precursor ion Product ion 1 CE 1 Product ion 2 CE 2 S‐lens (V) Azure B 4.74 270.1 212.0 48 254.1 31 100 Methylene Blue 4.77 284.1 252.0 53 268.1 33 100 Crystal Violet 4.93 374.2 239.0 33 358.2 28 100 Malachite Green 5.59 329.1 165.1 59 313.2 37 100 Nile Blue 5.76 318.1 245.1 55 274.1 35 100 Leucomalachite Green 5.98 331.1 223.1 50 239.1 29 100 Leucocrystal Violet 5.99 372.2 340.2 51 356.2 37 100 Brilliant Green 6.19 385.2 297.1 51 341.2 37 100 Victoria Blue 6.27 470.2 333.1 48 349.1 34 145

Accuracy & precision Data for 9 Dyes Spiked in Salmon Tissue (n=5) MG LMG CV LCV NB AB MB BG VB 1 ppb Sample 1 92.2 83.8 88.1 101.7 87.2 94.2 76.5 98.5 98.2 Sample 2 94.0 109.5 83.4 92.8 85.4 97.3 80.0 92.9 90.4 Sample 3 92.6 91.2 83.0 95.5 85.0 96.8 77.6 92.1 91.4 Sample 4 93.5 105.6 94.9 91.3 85.1 94.3 73.3 93.2 92.5 Sample 5 92.4 81.2 83.1 103.0 87.9 96.0 76.8 99.0 93.4 Mean 92.9 94.3 86.5 96.9 86.1 95.7 76.9 95.1 93.2 RSD 0.83 13.52 5.97 5.44 1.58 1.51 3.13 3.51 3.26

10 ppb Sample 1 97.1 91.1 92.6 100.4 95.9 96.6 85.6 106.9 104.4 Sample 2 99.7 96.6 99.1 105.9 98.6 98.6 81.8 103.2 104.4 Sample 3 98.7 94.9 98.3 97.6 97.5 99.4 87.0 104.2 102.7 Sample 4 99.9 88.1 91.0 107.3 99.1 97.9 88.2 98.5 92.2 Sample 5 100.0 97.5 98.6 97.4 99.5 93.5 86.5 102.1 104.3 Mean 99.1 93.7 95.9 101.7 98.1 97.2 85.8 103.0 101.6 RSD 1.24 4.24 3.99 4.56 1.49 2.39 2.85 2.98 5.21

RT: 0.00 - 7.50 SM : 5G RT: 0.00 - 7.50 SM : 5G 4.74 NL: 1.84E4 5.75 NL: 5.79E4 10 0 10 0 TIC F: + c ESI SRM ms2 TIC F: + c ESI SRM ms2 270.104 90 318.086 [ 2 12 . 0 19 - 2 12 . 0 2 1, [245.049-245.051, 80 254.069-254.071] MS 80 274.079-274.081] MS Azure B 1ppb_sample2 Nile Blue 1ppb_sample2 60 70 60 40 50

40 20 30 4.00 5.67 0 4.78 NL: 2.08E4 20 10 0 TIC F: + c ESI SRM ms2 10 284.069 [252.049-252.051, 0 80 5.98 NL: 3.68E3 268.089-268.091] MS 10 0 Methylene Blue 1ppb_sample2 m/z= 222.58-223.58 F: + c ESI SRM ms2 60 90 331.157 80 [223.079-223.081, 239.109-239.111] MS 40 Leucomalachite Green 70 1ppb_sample2

60 20 50 4.13 5.94 0 NL: 2.67E3 40 10 0

m/ z= 357.70-358.70 F: + Relative Abundance c ESI SRM ms2 30 374.222 80 20 [239.089-239.091, Crystal Violet 358.199-358.201] MS 10 60 1ppb_sample2 5.30 6.59 0 6.23 NL: 2.32E4 6.55 10 0 TIC F: + c ESI SRM ms2 40 90 385.185

Relative Abundance [297.119-297.121, 20 80 341.199-341.201] MS Brilliant Green 1ppb_sample2 70 0 5.63 N L: 1. 16 E 4 60 10 0 TIC F: + c ESI SRM ms2 329.141 50 [165.059-165.061, 80 313.159-313.161] MS 40 Malachite Green 1ppb_sample2 30 60 20

40 10

0 6.28 NL: 2.82E4 20 10 0 TIC F: + c ESI SRM ms2 90 470.231 0 [333.089-333.091, 6.01 NL: 3.79E4 349.119-349.121] MS 10 0 80 TIC F: + c ESI SRM ms2 Victoria Blue 1ppb_sample2 372.218 70 [340.149-340.151, 80 356.189-356.191] MS 60 Leucocrystal Violet 1ppb_sample2 50 60 40

40 30

20 20 10 5.37 0 0 0 2 4 6 0 2 4 6 Time (min) Time (min)

Figure 2. Chromatogram of salmon tissue fortified with the 9 dyes a 1 ng/g. RT: 0.00 - 7.49 SM : 5G RT: 0.00 - 7.49 SM : 5G 4.74 NL: 1.67E5 5.76 NL: 4.07E5 10 0 10 0 TIC F: + c ESI SRM TIC F: + c ESI SRM ms2 ms2 270.104 90 318.086 [212.019-212.021, [245.049-245.051, 80 254.069-254.071] 80 274.079-274.081] MS Azure B MS 10ppb_sample5 10ppb_sample5 Nile Blue 60 70 60 40 50

40 20 30 0 4.77 NL: 1.94E5 20 10 0 TIC F: + c ESI SRM 10 ms2 284.069 5.18 6.88 [252.049-252.051, 0 80 5.98 NL: 2.31E4 268.089-268.091] 10 0 Methylene Blue MS 10ppb_sample5 m/z= 222.58-223.58 F: + c ESI SRM ms2 331.157 60 90 [223.079-223.081, 80 239.109-239.111] MS 10ppb_sample5 40 70 Leucomalachite Green

60 20 50 4.13 0 NL: 3.56E4 40 10 0

TIC F: + c ESI SRM Relative Abundance ms2 374.222 30 [239.089-239.091, 80 20 358.199-358.201] Crystal Violet MS 10ppb_sample5 10 60 5.31 6.79 0 6.20 NL: 2.04E5 10 0 TIC F: + c ESI SRM ms2 40 90 385.185

Relative Abundance [ 297.119-297.121, 20 80 341.199-341.201] MS 5.98 Brilliant Green 10ppb_sample5 70 0 5.59 NL: 1.26E5 60 10 0 TIC F: + c ESI SRM ms2 329.141 50 [165.059-165.061, 80 313.159-313.161] 40 Malachite Green MS 10ppb_sample5 30 60 20

40 10

0 6.27 NL: 1.98E5 20 10 0 TIC F: + c ESI SRM ms2 470.231 6.68 90 0 [333.089-333.091, 5.99 NL: 2.97E5 349.119-349.121] MS 10 0 80 TIC F: + c ESI SRM Victoria Blue 10ppb_sample5 ms2 372.218 70 [340.149-340.151, 80 356.189-356.191] 60 Leucocrystal Violet MS 10ppb_sample5 50 60 40

40 30

20 20 10

0 0 0 1 2 3 4 5 6 7 0 2 4 6 Time (min) Time (min)

Figure 3. Chromatogram of salmon tissue fortified with the 9 dyes a 10 ng/g.

Malachite_Green Y = 13494.3+95258.7*X R^2 = 0.9991 W: 1/X

2000000

1800000

1600000

1400000

1200000

Area 1000000

800000

600000

400000

200000

0 0 2 4 6 8 10 12 14 16 18 20 22 ng/ml

Figure 4. Calibration curve example (malachite green).

* TMPD: N,N,N’,N’‐tetramethyl‐1,4‐phenylenediamine dihydrochloride

**Mcilvaines buffer pH 3.5 – mix 500mL of 0.1M citric acid with 400mL 0.1M disodium hydrogen phosphate. Adjust pH to the correct value (3.5) using the citrate or phosphate solution.

DCN-311011-290