[Technical Report] Simultaneous Analysis of Veterinary Drugs Using STQ Method

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Aug, 2021

This article was written by Ms. Mikie Shima, AiSTI SCIENCE Co., Ltd., Technical & Marketing Group, for Vol. 13 (August 2020), of ChemGrowing.
The content of this article is from the time of publication. It is not the latest information due to new knowledge and changes in regulatory rules after original publication.

Introduction

As interest in food security and safety is increasing, the analysis of veterinary drugs remaining in food is important. However, there are various kinds of veterinary drugs with different physical properties, and it is not easy to analyze them simultaneously. In addition to analytical precision, "speed" and "simplicity" are also required. In this application, we introduce the results obtained by applying the STQ (Solid phase extraction Technique with QuEChERS method) method, which is a "rapid, simple, and highly purified" method for analysis of residual pesticides, to the analysis of veterinary drugs.

Outline of STQ Method

The outline of STQ method is shown in Figure 1.

In the STQ method, extraction of a sample homogenized by the pre-freezing and cryogenic milling with dry ice method is performed referring to the QuEChERS method, and the sample is purified using a solid phase column Smart-SPE with a small filling amount of several tens of mg.

Figure 1. Outline of STQ Method

In the pre-freezing and cryogenic milling with dry ice method, homogenization is assured even with a sample collected in a small amount because the samples can be ground to a fine powder by the method. Then, downscaling the analytical system is attempted by combining extraction by the QuEChERS method with solid-phase column Smart-SPE. Thus the analysis can be done more rapidly, with improved operability and lower cost.

In the purification process of the STQ method, a high purification effect is achieved by using a "solid-phase column." Because the extract is prepared by the QuEChERS method, which is "partitioning-only," purification is insufficient and pesticides are also adsorbed. On the other hand, the solid-phase column extraction technique adopted in the STQ method enables efficient recovery of pesticides while adsorbing impurities in the "partition/separation action" level. The operability of a solid phase column is simplified and streamlined by using the dedicated fully automated solid-phase extractor system ST-L400 and pretreatment kit. As the purification affects the reliability of the qualitative and quantitative analysis of peaks and the frequency of maintenance of the measuring device, improvement of the purification is one of the most important factors in pretreatment.

As described above, the STQ method is a "rapid, simple, and highly purified" method that takes into account the total balance from homogenization of samples to analysis.

Analytical Method

Analytical Samples

In this application, chicken tender, pork medallion and beef fillet were used as meat (muscle), chicken egg and milk were used as other stock farm and aquatic products, and frozen deep-fried chicken as an example of processed meat products. All of them were commercial products.

Standard Solutions

The following mixture standard solutions manufactured by FUJIFILM Wako were used.

Veterinary drug mixture standard solutions (quinolones) (Code No. 227-02051)
Veterinary drug mixture standard solutions (sulfa drugs + antifolates) (Code No. 228-02081)
Veterinary drug mixture standard solutions (macrolides) (Code No. 225-02091)
Veterinary drug mixture standard solutions (hormones) (Code No. 220-02203)

Preparation of Samples

Homogenization of samples is important for maintaining analytical precision. The same is true for meat samples, but in the case of room temperature milling, fibers may remain intact or the samples may become lumpy and not be uniformly milled. Using the pre-freezing and cryogenic milling with dry ice method, the samples are pre-frozen with dry ice and then ground with dry ice in the frozen state to be homogenized into a powder (Figure 2). In the present recovery test, chicken tender, pork medallion, beef fillet and frozen deep-fried chicken were ground by the pre-freezing and cryogenic milling with dry ice method using a Large Volume Freeze Crusher (Code No. 387-17711) (Figure 3).

Whole chicken eggs were agitated in the food processor to homogenize.

Figure 2. Comparison between Milling at Room Temperature and Pre-freezing and Cryogenic Milling with Dry Ice (Chicken Tender)

Figure 3. Large Volume Freeze Crusher

Extraction

Quinolones used in the recovery test are compounds that have a basic skeleton of 4-quinolone and contain a carboxyl group (-COOH) in their structure. This highly polarized group becomes dissociated under neutral conditions and exhibits low polarity in acidic condition (Figure 4).

Figure 4. Quinolones

First, components other than quinolones are extracted using acetonitrile (neutral) and transferred to the acetonitrile layer. Since quinolones are dissociated at this time, they move to the aqueous layer. Then, acidic acetonitrile containing formic acid is added to the residue, and undissociated quinolones are extracted and transferred to the acetonitrile layer containing formic acid Then both extracts are combined and made to a constant volume. As described above, it is possible to transfer the quinolones, which are contained in the aqueous layer in the neutral condition, to the solvent layer by the "constant volume method with repetitive extraction" using neutral acetonitrile and acidic acetonitrile made with formic acid, and combining this with other components by a constant volume operation to ensure precision quantitatively (Figure 5).

Figure 5. Constant Volume Method with Repetitive Extractions

After weighing 5 g of each sample, the standard solution in 3-2 was added to make the concentration of each sample 0.01 ppm. Then, water was added to make the water content in the samples 5 g for chicken tender, pork medallion, beef fillet and frozen deep-fried chicken in which the water content is less than 80%. After addition of 10 mL of acetonitrile, the samples were homogenized for 1 minute. 0.5 g of sodium chloride and 2 g of anhydrous magnesium sulfate were added, and the mixture was shaken vigorously by hand for 1 minute. After centrifugation, the acetonitrile layer ([1]) was transferred to a graduated test tube. 10 mL of acetonitrile containing 5% formic acid was added to extract quinolones. Homogenization was performed for 1 minute, followed by vigorous shaking by hand for 1 minute. After centrifugation, 5% formic acid-containing acetonitrile layer ([2]) was transferred to the graduated test tube and combined with [1]. The volume was made 20 mL with acetonitrile and the solution was used as the extract (Figure 6).

Figure 6. Pretreatment Flow

Purification

Purification was performed by the fully automated solid phase extraction system ST-L400 (Figure 7) using a total of 3 solid-phase columns comprising of Smart-SPE C18-50 (Code No. 304-94981), C18-30 (Code No. 307-94971) and PSA-30 (Code No. 301-94991).

Figure 7. Fully Automated Solid-phase Extraction System ST-L400 and Smart-SPE Solid-phase Column

In the fully automated solid-phase extraction system ST-L400, each solid-phase column is connected as shown in Figure 8 and the solution is passed through the columns. C18 is a non-polar solid phase column and retains non-polar and low-polar components in the sample. PSA retains acidic and ionic substances. In Figure 8, [1] C18-50 retains non- and low-polar impurities after loading the sample allowing passage of target components.

In [2] PSA-30 connected beneath C18-50, acidic substances such as fatty acids and ionic impurities are retained and pass through the target components. Since quinolones are retained in addition to these impurities, an acidic solution is passed through after loading the sample to elute undissociated quinolones from PSA. To the effluents from these two types of connected solid-phase columns, water is added from the nozzle connected beneath the solid-phase column [1] and [2] to lower the ratio of acetonitrile, and the solution is passed through [3] C18-30. Water is added to the effluent to reduce the ratio of acetonitrile and low-polar impurities that were not eliminated with [1] C18-50 are eliminated with [3] C18-30. Above operations can reduce the burden of impurities to the analytical columns (ODS).

Figure 8. Purification Process with Fully Automated Solid-phase Extraction System ST-L400

1 mL of the extract obtained in 3-4. was loaded to the port of C18-50 (50 mg) and PSA-30 (30 mg), and 0.5 mL of 2% formic acid-containing acetonitrile-water mixture (8/2) was passed through the column. The effluents from C18-50 and PSA-30 were passed through C18-30 (30 mg) while adding 0.5 mL of water. The effluents were all received in a test tube and the volume was increased to 2 mL (Figure 6).

In order to confirm the influence of ionization inhibition at the time of measurement, the sample (without the addition of the standard solution) was pretreated in the same manner as the above, purified and added standard solution to make the concentration 0.01 ppm in the sample.

Measuring Conditions

The samples obtained were measured by LC-MS/MS. The measurement conditions are shown in Table 1.

Table 1. Measuring Conditions

Instrument
LC	: Nexera X2 (SHIMADZU Co.)
LC-MS	: LCMS-8045 (SHIMADZU Co.)
LC conditions
Column	: YMC-Triart C18 (150 x 2.1 mm I.D., S-3 μm, 12 nm)
Eluent	: Mobile phase　A: 0.1% formic acid in water B: 0.1% formic acid in acetonitrile
Flow rate	: 0.2 mL/min
Gradient	: B Conc. 1% (0 min)→15% (1 min)→40% (6 min)→100% (10-15 min)→1% (15.01-18 min
Injection vol.	: 5 μL (with water 10 μL）
Column temperature	: 40℃
MS conditions
Ionization	: ESI positive and negative
Mode	: MRM

Results

The results of the recovery test of the 46 components in the mixture standard solutions are shown in Table 2, and representative chromatograms are shown in Figure 9. The recovery rate was calculated by comparing the peak area of each component in the recovery test sample with that from the standard solution at the same concentration. The concentration of the recovery test samples in the vial was 1.25 ppb. In addition, the following 2 types of recovery rates were calculated to confirm the influence of ionization inhibition during measurement.

A: Recovery rate (%) with the use of solvent std

Solvent std

Mixture standard solution prepared by diluting the standard solution in 3-2. to 1.25 ppb with the solvent (the diluent is a mixture of acetonitrile, 5% formic acid-containing acetonitrile, water, and 2% formic acid-containing acetonitrile-water (8/2) in 1:1:1:1)

Calculation formula

Area from the recovery sample / area from 1.25 ppb of the solvent std x 100

B: Recovery rate (%) with the use of matrix std

Matrix std

The solution with the addition of standard after purification in 3-5. (concentration in the vial is 1.25 ppb)

Calculation formula

Area from the recovery sample / area from 1.25 ppb of the matrix std x 100

Figure 9. Representative Chromatograms

Table 2. Recovery Test Results

Spiked concentration: 0.01 ppm in the sample (n=3)

No.	Compound name	Classi fication¹⁾	Ionization mode²⁾	Retention time³⁾ (minutes)	Chicken tender		Pork medallion		Meef fillet		Milk		Egg		Deep-fried chicken
					When solvent std is used⁴⁾	When matrix std is used⁵⁾	When solvent std is used⁴⁾	When matrix std is used⁵⁾	When solvent std is used⁴⁾	When matrix std is used⁵⁾	When solvent std is used⁴⁾	When matrix std is used⁵⁾	When solvent std is used⁴⁾	When matrix std is used⁵⁾	When solvent std is used⁴⁾	When matrix std is used⁵⁾
					Recovery (%)	Recovery (%)	Recovery (%)	Recovery (%)	Recovery (%)	Recovery (%)	Recovery (%)	Recovery (%)	Recovery (%)	Recovery (%)	Recovery (%)	Recovery (%)
1	alpha-Trenbolone	H	P	10.108	95	92	119	100	83	83	96	89	85	87	82	111
2	beta-Trenbolone	H	P	10.110	106	92	88	95	94	86	100	111	101	100	77	85
3	Ciprofloxacin	Q	P	5.912	72	63	70	72	65	70	74	77	73	70	68	94
4	Clostebol	H	P	11.337	86	94	87	95	84	90	87	86	74	85	87	96
5	Danofloxacin	Q	P	6.064	76	72	75	82	74	68	71	82	67	80	57	83
6	Diaveridine	S	P	5.486	69	94	69	96	59	91	70	87	84	85	61	104
7	Difloxacin	Q	P	6.969	88	80	85	81	77	73	86	90	67	80	85	85
8	Enrofloxacin	Q	P	6.308	81	83	78	85	79	75	103	87	82	69	75	92
9	Flumequine	Q	P	9.880	88	84	85	93	87	84	91	85	77	74	90	88
10	Marbofloxacin	Q	P	5.661	89	85	80	78	78	74	81	83	77	79	100	86
11	Methylprednisolone	H	P	9.435	107	102	88	109	88	78	97	87	112	83	91	82
12	Nalidixic Acid	Q	P	9.809	87	85	84	91	87	82	89	87	74	77	105	91
13	Norfloxacin	Q	P	5.762	74	65	69	70	59	48	84	75	75	76	72	93
14	Ofloxacin	Q	P	5.820	73	74	85	74	75	74	83	79	82	72	81	88
15	Orbifloxacin	Q	P	6.524	79	79	80	71	75	73	85	83	73	67	78	82
16	Ormetoprim	S	P	6.081	88	96	83	96	79	86	79	91	88	87	76	95
17	Oxolinic Acid	Q	P	8.866	86	93	82	98	88	82	88	96	77	82	63	93
18	Piromidic acid	Q	P	10.405	84	84	89	79	82	76	86	84	74	68	88	87
19	Prednisolone	H	P	8.941	92	107	92	92	93	89	99	97	89	95	69	95
20	Pyrimethamine	S	P	7.715	87	94	83	99	84	89	90	87	98	91	109	99
21	Sarafloxacin	Q	P	6.846	92	75	74	74	65	62	91	81	77	62	85	77
22	Sulfabenzamide	S	P	9.120	101	97	98	99	85	82	87	89	78	89	91	95
23	Sulfabromomethazine Na	S	P	9.898	99	98	108	111	87	91	91	97	87	98	84	87
24	Sulfacetamide	S	P	5.747	98	105	87	95	92	102	96	89	88	89	76	103
25	Sulfachlorpyridazine	S	P	8.119	96	94	99	94	92	88	93	96	80	95	98	98
26	Sulfadiazine	S	P	6.056	92	94	86	93	78	90	81	91	88	88	57	96
27	Sulfadimethoxine	S	P	9.171	105	105	89	100	91	93	92	92	91	86	94	96
28	Sulfadimidine	S	P	7.234	98	99	93	100	88	85	89	86	98	91	92	98
29	Sulfadoxine	S	P	8.429	98	98	90	105	93	92	91	89	91	89	91	96
30	Sulfaethoxypyridazine	S	P	8.398	94	99	84	99	88	82	90	94	89	91	100	99
31	Sulfamerazine	S	P	6.718	97	95	81	98	88	98	109	94	91	88	77	96
32	Sulfamethoxazole	S	P	8.472	99	103	88	99	93	105	85	84	96	90	91	93
33	Sulfamethoxypyridazine	S	P	7.165	98	102	90	99	87	86	88	92	89	104	92	100
34	Sulfamonomethoxine	S	P	7.726	96	92	93	100	90	88	96	98	89	90	99	95
35	Sulfanitran	S	N	9.736	108	104	108	93	103	91	98	96	101	86	110	92
36	Sulfapyridine	S	P	6.326	92	92	90	96	83	85	69	91	82	96	69	99
37	Sulfaquinoxaline	S	P	9.154	97	98	88	98	88	88	90	86	97	90	98	97
38	Sulfathiazole	S	P	6.047	88	94	85	99	72	76	84	91	76	90	61	94
39	Sulfatroxazole	S	P	8.597	93	87	100	105	99	95	89	87	92	90	79	95
40	Sulfisomidine	S	P	5.250	79	91	85	96	80	84	79	88	81	92	65	96
42	Sulfisozole	S	P	7.600	96	101	88	93	91	89	86	90	86	98	78	96
43	Tiamulin	M	P	9.210	99	98	95	99	86	89	89	89	96	95	79	97
44	Tilmicosin (isomers)	M	P	7.629	98	100	92	99	86	88	82	86	91	96	105	96
45	Trimethoprim	S	P	5.751	79	98	87	96	91	93	80	93	78	91	114	105
46	Zeranol	H	P	10.340	80	105	119	98	119	106	96	89	95	94	107	119

Q: quinolones , S: sulfa drugs or antifolates, M: macrolides, H: hormones
Ionization mode, P: Positive, N: Negative
Measured values by AiSTI SCIENCE Co., Ltd.
Recovery sample / solvent std x 100
Recovery sample / matrix std x 100

Good recovery rates of 70 to 120% were obtained for 42 or more components in 3 types of meat, milk and chicken egg. With the frozen deep-fried chicken, the recovery rate of 70 to 120% was given by 39 components but it was less than 70% in the remaining 7 components. By calculating the recovery rate as the matrix std, the recovery rate was 70 to 120% in each component. This is probably because the samples recovered by the purification process were affected by ionization inhibition of matrix during measurement.

The recovery rate was about 70% for some components of quinolones. It was assumed that these substances were not sufficiently transferred to the solvent layer during extraction. In addition, this trend was prominent with beef, suggesting the influence of the difference in impurity composition in the samples.

Conclusions

By repetitive extractions with two types of solvents, acetonitrile and 5% formic acid-containing acetonitrile and adding up the extracts, components with different physical characteristics could be pretreated at the same time. In addition, quantitativeness could be ensured by adding up the two extracts and then doing constant volume.

In purification process, by using the dedicated fully automated solid-phase extractor system ST-L400, the complicated process could be performed quickly and easily.

In this recovery test, not only meat but also milk, chicken eggs, and processed foods (deep-fried chicken) were able to obtain generally good results with a uniform standard concentration.

In this study, we proposed pretreatment methods for veterinary drugs, and demonstrated that they could be analyzed quickly, easily, and in highly purified manner for the respective samples.

References

Shima M. et al.: Proceedings of the 114th Meeting of the Japanese Society for Food Hygiene and Safety, p.124 (2018).
Shima M. et al.: Proceedings of the 115th Meeting of the Japanese Society for Food Hygiene and Safety, p.110 (2019).

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