Quinoxaline drug residue analysis technology-extraction method

The analysis objects of quinoxaline residues mainly include the original form of the drug and its metabolites
.

In addition, quinoxaline drugs in animal tissues are metabolized by the body and deoxygenated at the N1 and N4 positions in the animal body

15.
1.
4.
1 Pre-processing method

(1) Extraction method

Quinoxaline drugs are not suitable for extraction under high temperature and high pressure.
At present, traditional liquid liquid extraction (LLE) is mainly used to extract such drugs from animal-derived matrices
.

The original drugs such as CBX and OLQ in this class of drugs and their deoxygenation and deoxygenation and deoxygenation: the deoxygenated metabolites corresponding to dioxygen are not combined with the tissues and can be directly extracted

1) Direct extraction

The original form of the drug and the intermediate deoxygenated metabolites are not combined with the tissues and can be directly extracted
.

Huang et al.
determined CYX and its metabolite BDCYX in chicken blood, muscle, liver, kidney and fat
.

The plasma was extracted with an equal volume of methanol by vortexing for 2 minutes; for other tissues, 2g samples were first added with 1 mL of water, and after vortexing for 1 minute, the fat samples were extracted with methanol - acetonitrile (1+1, v/v), and other samples were extracted with acetic acid Ethyl ester extraction, 4mL for the first time, followed by 2×2mL extraction, vortex mixing, ultrasonic extraction for 5 minutes, low-temperature centrifugation to take the organic solvent layer, clean up and use high performance liquid chromatography (HPLC) to determine

Aerts et al.
used methanol-acetonitrile to directly extract CBX and its metabolites 1-deoxycarbaoxide, 4-deoxycarbaoxide and BDCBX from pig tissues
.

Add 40 mL methanol-acetonitrile (1+1, v/v) to 10 g homogenized samples (muscle, liver, kidney), shake and mix for 15 minutes, centrifuge at 2000 g for 5 minutes, and take the supernatant for further purification

Huang et al.
determined QCT and its metabolite BDQCT in pig and chicken tissues, 5g muscle/liver plus 2mL water, 15mL ethyl acetate; 5g fat plus 15mL methanol-acetonitrile (1+1, v/v), shaking for 1min, ultrasound for 1min After centrifugation, the supernatant was evaporated to dryness, the residue was dissolved in acetonitrile and isooctane and liquid-liquid partitioning, the benzalkonium layer was evaporated, reconstituted, and then analyzed by HPLC
.

LOD and LOQ were 0.

Zhang et al.
determined MQX and its four metabolites in swine urine: 1-deoxymethequine, 4-deoxymethequine, BDMQX and MQCA
.

Add 200μL of methanol and 60μL of tetrachloroethane to the 5mL sample , ultrasonically extract for 4min, centrifuge and determine by HPLC

2) Extract after hydrolysis

A.
Extract after alkaline hydrolysis

The alkaline hydrolysis method is used to break the amide bond between QCA or MQCA and protein.
The alkaline hydrolysate is generally neutralized with acid and then extracted with an organic solvent.
The most commonly used is ethyl acetate
.

During the hydrolysis process, part of the protein in animal tissues is also hydrolyzed to produce small-molecule amino acids and peptides, which need to be purified of hydrolysis interferents

Huang et al.
determined the QCT metabolite MQCA in pig and chicken tissues.
The sample was hydrolyzed with 3mol/L sodium hydroxide at 95～100℃ for 30～40min, then neutralized with concentrated hydrochloric acid, and then extracted with ethyl acetate for 2min.
The ethyl acetate extract was further purified and determined by HPLC
.

The LOQ is 0.

Huang et al.
used 3mol/L sodium hydroxide solution at 100°C for 30 minutes to hydrolyze the CYX metabolite QCA in chicken blood and tissues.
The hydrolyzate was neutralized with hydrochloric acid, and then extracted with ethyl acetate.
QCA in the ethyl acetate extract It is then back-extracted to citric acid buffer, purified by a cation exchange column, and then purified by liquid-liquid partitioning with dilute hydrochloric acid and chloroform, and detected by HPLC
.
The LOQ of QCA is 0.
025mg/kg (plasma and visceral tissue) and 0.
02mg/kg (muscle); the recovery rate is 70% to 87%, and the daily RSD is 7.
69% to 1.
43%
.
Zhang; et al.
To determine the CYX metabolite QCA in sheep tissue, add 10mL 3mol/L sodium hydroxide solution to 5g sample, hydrolyze at 100℃ for 30min, cool to room temperature, add 4mL concentrated hydrochloric acid to mix evenly and neutralize, centrifuge, take the supernatant to dissociate After the solution is further purified, it is determined by HPLC
.
The recovery rate of QCA is 70%-82%, the daily CV is 6.
6%-11.
1%; the LOQ is 25μg/kg, and the LOD is 15μg/kg
.
Hutchinson et al.
measured CBX metabolite QCA in pig liver.
Add 10mL of 3mol/L sodium hydroxide solution to 5g tissue and hydrolyze the sample at 100℃ for 30min to dissociate the QCA bound to the tissue.
The hydrolyzate was fully neutralized with 4mL concentrated hydrochloric acid and acetic acid After ethyl ester extraction, the extract was purified by liquid-liquid partition and solid-phase extraction, and then determined by LC-MS/MS
.
The method recovery rate was 89%～109%, CV was 0.
15%～10%, CCa and CCβ were 0.
16 ug/kg and 0.
27 μg/kg, respectively
.

B.
Extract after acid hydrolysis

Hydrolysis of QCA and MQCA in blood and tissues with sodium hydroxide solution needs to be carried out at a high temperature of 90～100℃.
Some proteins in animal tissues will also be decomposed, which will produce more small molecule amino acids, which will increase the interference of sample determination and affect the detection.

.
Under acidic conditions, the hydrolysis mainly adopts dilute acid or dilute acid organic solvent mixture to hydrolyze.
The heating temperature is lower, the free matrix components produced by hydrolysis are less, and the acid hydrolysis is more thorough, reducing the degradation loss of MQCA and QCA, and effectively releasing the combined state.
The effect of MQCA and QCA is better than alkaline hydrolysis
.
After the drug is hydrolyzed, it is transferred to the acidic hydrolysis solution, and the hydrolysis solution is centrifuged during extraction, and the supernatant is directly taken for purification, so that the hydrolysis and extraction are performed simultaneously
.
The most commonly used acids are hydrochloric acid and metaphosphoric acid
.

Zhang et al.
established a method for the determination of residues of OLQ metabolism marker MQCA in fish and shrimp
.
Add 5g sample to 15mL2mol/L hydrochloric acid solution, vortex for 2min, shake and acid hydrolyze the lid at 60℃ for 1h, then centrifuge at 8000g for 10min, collect the acid hydrolysis supernatant, purify it by MAX cartridge, and determine by LC-MS/MS
.
The LOD and LOQ were 0.
1ng/g and 0.
25ng/g, respectively.
Within the range of 0.
25～50.
0ng/g, the average recovery rate was 92.
7%～104.
3%, and the RSD was less than 6%
.
Lv Hailuan et al.
133 used 0.
2mol/L hydrochloric acid to shake at 60℃ for 1h to hydrolyze and extract the residue marker MQCA of OLQ in pork, pig liver, pig kidney, fathead fish, prawn and crab tissue, and the supernatant was directly extracted with C18 solid phase extraction.
Column cleanup, LC-MS/MS determination
.
The LOD of MQCA in pork, pig liver, pig kidney, fish, prawn and crab are 0.
90μg/kg, 1.
51μg/kg, 0.
94μg/kg, 1.
04μg/kg, 1.
62μg/kg and 1.
80μg/kg, respectively, LOQ They are 3.
00μg/kg, 5.
02μg/kg, 3.
13μg/kg, 3.
46μg/kg, 5.
40μg/kg, 6.
00μg/kg; the average recovery rate of MQCA is between 73.
6% and 89.
0%, and the RSD within the day is between 73.
6% and 89.
0%.
15% or less, daytime RSD is less than 20%
.
Mei Jingliang et al.
established a residual analysis method for OLQ and its metabolite MQCA in carp and prawns
.
Take 2.
5g of tissue and add 7mL 0.
3mol/L HCI solution, shake and extract for 2min, centrifuge to transfer the supernatant, and then extract the residue with 6mL 0.
3mol/L HCI solution once, purify with _C18 solid phase extraction column, blow dry with nitrogen and use After constant volume of mobile phase, HPLC determination
.
When the addition levels of OLQ and MQCA in fish tissues were 20～200μg/kg and 35～200μg/kg, the average recovery rates were 83.
5%～87.
7% and 79.
6%～87.
4% respectively; when OLQ and MQCA were in shrimp tissues When the addition level of the two compounds is 15～200μg/kg and 30～200μg/kg, the average recovery rate is correspondingly 74.
6%～80.
9% and 81.
0%～86.
6%; the intra-assay RSD of the two compounds in the two tissues is 0.
51% ~ 4.
47%, inter-assay RSD of 0.
66% ~ 7.
58%; carp tissues OLQ and MQCA of LOD were 6ug / kg and 10ug / kg, LOQ were 20μg / kg and 35μg / kg; of
shrimp tissue OLQ and MQCA The LODs are 4.
5μg/kg and 8μg/kg, respectively, and the LOQs are 15μg/kg and 30ug/kg, respectively
.

Yong et al.
established a method for the determination of QCT metabolite MQCA in chicken tissues.
A 2g sample was first extracted with chloroform-acetonitrile (1+2, v/v) ultrasonic waves twice, each time was ultrasonically extracted for 10 minutes, and then transferred twice after centrifugation.
Add 1mol/L hydrochloric acid to the tissue and hydrolyze it at 90s℃ for 1 hour to dissociate the drug residues bound to the tissue.
After placing it at room temperature, add 10mL chloroform-acetonitrile (1+2, v/v) for ultrasonic extraction for 5 minutes, and centrifuge , Take the organic layer and evaporate to dryness, after further purification, LC-MS/MS determination
.
The method recovery rate is 77.
1%-95.
2%, CV is less than 15%; CCa is 0.
24-0.
76μg/kg, and CCβ is less than 2.
34μg/kg
.

The use of metaphosphoric acid for hydrolysis avoids the use of strong acids and bases, and the hydrolysis solution is directly extracted with organic solvents, which can simplify the operation
.
At the same time, metaphosphoric acid can also effectively precipitate tissue proteins, and the interference is significantly less than that of alkaline hydrolysis methods
.

Horie et al.
used 0.
3% methanol metaphosphate (7+3, v/v) to dissociate and extract CBX and its metabolites QCA and BDCBX from pig muscle and liver at the same time, vortex and mix, shake and extract for 10 minutes, centrifuge, and supernatant After purification by HLB solid phase extraction column, LC-MS/MS determination
.
When the added concentration is 2.
5ng/g and 5ng/g, the recovery rate of QCA and BDCBX is 70.
2%-86.
3%, and the LOD is 1ng/g
.
Xu et al.
determined the CYX metabolite QCA in pig liver and extracted it with metaphosphoric acid～methanol-water (2+20+78, v/v).
The recovery rate of LC-MS/MS determination was only 40%.
The reason was that metaphosphoric acid was deproteinized.
Incomplete, QCA re-adsorption resulted in a low recovery rate; instead of using 5% metaphosphoric acid-methanol (8+2, v/v) extraction, the recovery rate increased to 70%
.
Della et al.
determined QCA in pig liver, added metaphosphoric acid-methanol-water (2+20+78, v/v) solution to the sample, vortexed and extracted for 1 min, centrifuged to take the supernatant, and purified by gas chromatography-mass spectrometry (GC- MS) determination
.
The method recovery rate was 90%～102%, CV was 1.
7%～8.
4%, CCa and CCβ were 32μg/kg and 34ug/kg, respectively, LOD was 0.
2μg/kg, LOQ was 0.
7μg/kg
.

Sniegocki et al.
determined CBX and its metabolites BDCBX, QCA and OLQ and its metabolites MQCA in pig muscle.
5g sample was homogenized, and 6mL 2% metaphosphoric acid-20% methanol-water solution was added, mixed and extracted by ultrasound for 15min, then centrifuged at low temperature , Take the supernatant for further purification, and determine by LC-MS/MS
.
Method CCa is 1.
04～2.
11ug/kg, CCβ is 1.
46～2.
89ug/kg, and the recovery rate is 99.
8%～101.
2%
.
Wu et al.
extracted QCA and MQCA from animal tissues and fish meat, added 5% metaphosphoric acid-10% methanol solution, vortexed and extracted for 2 minutes, centrifuged, took the supernatant, repeated the extraction once, combined the two extracts, and further purified.
HPLC determination
.
CC of MQCA and QCA
.
It is 0.
7～2.
6μg/kg, CCβ is 1.
3～5.
6ug/kg, recovery rate is 70%～110%, RSD is less than 20%
.
Studies have found that adding a proper amount of methanol to the extraction solvent can improve the solubility of the drug, but when the methanol content exceeds 10%, the recovery rate is lower than 60%.
The reason may be that the excessive methanol content affects protein precipitation and reduces the solubility of metaphosphoric acid.
And affects the release of the tissue protein to the drug, thereby reducing the extraction recovery rate
.
Experiments show that 5% metaphosphoric acid and 10% methanol solution is the best
.

C.
Extract after enzymolysis

Enzymatic hydrolysis has more advantages than acid/alkali hydrolysis.
It can selectively destroy the bond between the drug and the tissue and reduce the co-extraction of the sample matrix, but it takes a long time, generally more than 16h
.
Before enzymolysis, formic acid can be used to digest and inactivate the tissue active enzymes of the matrix to improve the efficiency of enzymolysis
.
The sample after enzymolysis is then extracted with an acidic solvent, and the extraction also plays a role of deproteinization
.

Hutchinson et al.
determined the metabolites MQCA and QCA of OLQ and CBX in pig liver, adding 8 mL of 0.
2mol/L Tris/HCl buffer (pH 9.
6) and 50 μL of protease (protease type XIV, 50 mg/mL) to a 5 g homogenized sample.
After mixing, enzymolyze at 55°C overnight.
After the proteolysis solution is placed at room temperature, 1 mL of concentrated hydrochloric acid is added for acidification extraction, mixing, and centrifugation to obtain the supernatant.
After purification, it is determined by LC-MS/MS
.
The CCa and CCβ of QCA are 0.
4μg/kg and 1.
2μg/kg, respectively, and MQCA are 0.
7μg/kg and 3.
6μg/kg
.
Merou et al.
determined QCA and MQCA in pig, bovine muscle and pig liver, adding QCA-d4 internal standard to 5g sample, 10mL 0.
1mol/L Tris buffer (pH9.
5, containing 5mg subtilisin A), and incubating at 52℃ After 2h enzymatic hydrolysis, the supernatant of enzymatic hydrolysis was purified by a solid phase extraction column and determined by LC-MS/MS
.
The CCa and CCβ ranges of CBX, MQCA and QCA are 0.
09～0.
24μg/kg and 0.
12～0.
41μg/kg, respectively, the recovery rate is 92%～101% (MQCA, QCA) and 60%～62% (CBX), CV Less than 12%
.
Lin Li et al.
established an LC-MS/MS method for the residues of CBX and OLQ metabolites QCA and MQCA in milk and milk powder
.
Add 10mL 0.
6% formic acid solution to 5g milk sample, mix well and shake at 47℃ for 1h, then add 3mL 1.
0mol/L Tris solution and 0.
3mL protease aqueous solution, mix well, enzymolysis at 47℃ for 16-18h, after enzymolysis Add 20mL 0.
3mol/LHCI to the sample solution, shake and mix, centrifuge for 15min, filter the supernatant, and determine after purification by a solid phase extraction column
.
The LOQ of QCA and MQCA in milk is 0.
5 μg/kg, and that of milk powder is 4.
0 μg/kg; the average recovery rate is 68.
2%～82.
5%, and the CV is 3.
4%～12%
.
Liu Zhengcai and others determined the OLQ metabolic residue marker MQCA residues in animal-source foods.
5g homogeneous tissue samples were added to 8mL protein complex digestion solution (0.
2 mol/L Tris buffer salt solution containing 0.
1mol/L calcium chloride, HCI solution was adjusted to pH 9.
6), mix well, then add 0.
3mL 10g/L protease solution, mix well, enzymolyze in a shaker at 47°C for 16-18h, after the enzymatic hydrolysate is purified by solid phase extraction, it is determined by LC-MS/MS
.
The LOQ of MQCA in the sample is 0.
3～0.
5μg/kg, the average recovery is between 60.
7%～107%, and the RSD is between 4.
59%～14.
9%
.

Boison et al.
used 0.
6% formic acid at 47℃ for 1 hour to digest and inactivate active enzymes in pig muscle and liver, then added 1 mol/L Tris solution for neutralization, and then added 1 mL of protease to enzymatically digest at 47℃ for 16～18 h, then add 0.
3 mol/L hydrochloric acid and shake at high speed for 5 min to extract, centrifuge to take the supernatant for further purification by solid phase extraction, and determine by LC-MS/MS
.
The LOQs of BDCBX and QCA were 0.
05 μg/kg and 0.
5 μg/kg, and the LODs were 0.
025 μg/kg and 0.
3 μgkg, respectively, and the recovery rate was between 80% and 120%
.
The Chinese national standard GB/T 22984-2008 determines the residual amount of CBX and OLQ metabolites in milk and milk powder.
5g sample is mixed with 10 mL 0.
6% formic acid solution and shaken at 47°C for 1 hour, then 3 mL 1 mol/L Tris solution is added, and then Add 0.
3 mL of 0.
01 g/mL SIGMA P5147 protease, mix well and shake at 47°C for 16-18h
.
After enzymatic hydrolysis, use 0.
1 mol/L EDTA-Mcllvaine buffer solution (pH 4) for direct homogenization extraction and determination by LC-MS/MS
.
QCA and MQCA recovery is 75% ~ 88%, LOD was 0.
5μg / kg (milk) and 4μg / kg (milk
powder)
.
Studies have found that temperature and pH have a great influence on the biological activity of the enzyme, and the protease has the strongest activity at pH 8.
5 and 47°C
.
Before enzymatic hydrolysis, add 0.
6% (volume fraction) formic acid solution to the sample and place it in an air bath at 47°C for 1 hour to inactivate other types of enzymes naturally present in milk and milk powder, and then adjust with Tris buffer solution pH, add protease for enzymatic hydrolysis
.
Experiments have shown that this method is more effective than hydrolysis with formic acid and extraction with acetonitrile-water (1+1, v/v).
Not only is the reaction conditions mild, but the drug is separated from dairy products more thoroughly after enzymatic hydrolysis
.

Related link: Maximum allowable residue limits for quinoxaline drugs