Quinolone drug detection method for eliminating false positive of pefloxacin

Abstract

The invention belongs to the technical field of drug analysis, and provides a quinolone drug detection method for eliminating pefloxacin false positive. Compared with a previous quinolone drug detection method, the detection method has higher selectivity on pefloxacin, the liquid chromatography-low resolution tandem triple quadrupole mass spectrometry is adopted to detect aquatic products, m / z 219 is used as quantitative ions under certain collision energy, and the detection result is accurate and accurate. Whether a sample contains pefloxacin or not can be quickly and accurately determined, false positive results are eliminated, and the method is simpler, more economical, more efficient and more accurate.

Description

Technical Field

[0001] The invention relates to the technical field of drug analysis, and in particular to a quinolone drug detection method for eliminating pefloxacin false positives. Background Art

[0002] Quinolones are widely used in aquaculture due to their broad antimicrobial spectrum and low price. According to the sampling inspection conducted by the Chinese agricultural product market supervision department, quinolones have a high detection rate in aquatic products, so they are a routine indicator in the monitoring of veterinary drug residues in aquatic products. Most countries have clear regulations on the maximum residue of quinolones in food. For example, the EU and my country both stipulate that the maximum residue of enrofloxacin and ciprofloxacin in aquatic products is 0.1 mg / kg.

[0003] In terms of testing standards and economic costs, triple quadrupole mass spectrometers are currently the conventional instruments used to confirm the presence of the test substance in samples in most food safety testing laboratories. However, due to the low resolution of triple quadrupole mass spectrometers, co-eluted impurities in complex food matrices are prone to interference, thus affecting the reliability of the results. In recent years, there have been reports of some false negatives and false positives detected using triple quadrupole mass spectrometers. Liquid mass spectrometry is commonly used for the detection of quinolones in aquatic products, and the judgment is based on some current standards of the Ministry of Agriculture and Rural Affairs.

[0004] Recently, in the monitoring of quinolone drug residues in sea cucumbers using triple quadrupole mass spectrometry, it was found that the suspected ingredient pefloxacin was detected in the sample. Its precursor ion, two product ions and ion abundance ratio all met the standard requirements, and the retention time had a slight drift. Compared with the pefloxacin standard, the deviation was 0.233%, which was less than the 1% specified in the standard. It was initially determined to be pefloxacin. However, through a series of tests, it was confirmed that it was not pefloxacin, that is, a false positive result of pefloxacin occurred. In order to quickly and accurately confirm whether the sample contains pefloxacin and eliminate false positive results, it is necessary to develop a simpler, more economical, efficient and accurate (eliminating false positive results of pefloxacin) quinolone drug detection method. Summary of the invention

[0005] In view of the existing needs, the present invention provides a quinolone drug detection method that eliminates the false positive of pefloxacin. Liquid chromatography-low-resolution triple quadrupole mass spectrometry is used to detect sea cucumber samples, and m / z 219 is used as a quantitative ion. This not only improves the accuracy of quinolone drug detection and eliminates the false positive of pefloxacin, but also does not require other methods for further verification and coordination, nor does it require the use of a high-cost high-resolution mass spectrometer, and can quickly confirm.

[0006] Specifically, the present invention provides a quinolone drug detection method for eliminating pefloxacin false positives, using liquid chromatography-low resolution triple quadrupole mass spectrometry to detect quinolone drug residues in sea cucumber samples, and using m / z 219 as a quantitative ion to confirm whether the sample contains pefloxacin.

[0007] In some embodiments, the abundance ratio of m / z 290 to m / z 316 is 40%-50%, which further confirms whether the sample contains pefloxacin.

[0008] In some embodiments, the abundance ratio of m / z 233 to m / z 290 is 20%-30%, which further confirms whether the sample contains pefloxacin.

[0009] In some embodiments, the detection conditions of the liquid chromatography are:

[0010] Chromatographic column: C18 column, 2.1mm×150mm, 5μm;

[0011] Flow rate 0.2-0.3mL / min;

[0012] Gradient elution: mobile phase A: 0.1% (V / V) formic acid-water solution, 5 mM buffer salt; mobile phase B: methanol or acetonitrile; elution program: 0-3.0 min, 15%-40% mobile phase B; 3.0-9.0 min, 40% mobile phase B; 9.0-10.0 min, 40%-15% mobile phase B; 10.0-15.0 min, 15% mobile phase B.

[0013] In some embodiments, the chromatographic column: CAPCELL PAK C18 MGⅡ chromatographic column, 2.1 mm×150 mm, 5 μm;

[0014] Flow rate: 0.25 mL / min;

[0015] Gradient elution: mobile phase A: 0.1% (V / V) formic acid-water solution, 5 mM ammonium acetate; mobile phase B: methanol; elution program: 0-3.0 min, 15%-40% mobile phase B; 3.0-9.0 min, 40% mobile phase B; 9.0-10.0 min, 40%-15% mobile phase B; 10.0-15.0 min, 15% mobile phase B.

[0016] In some embodiments, the low resolution triple quadrupole mass spectrometry detection conditions are as follows:

[0017] Ionization mode: electrospray ESI ion source positive mode;

[0018] Spray voltage: 4000V;

[0019] Sheath gas flow: 35.0arb;

[0020] Auxiliary gas: 10.0arb;

[0021] Capillary temperature: 350℃;

[0022] Collision gas: argon;

[0023] Scanning mode: multiple reaction monitoring (SRM) mode.

[0024] In some embodiments, the sea cucumber sample is pretreated:

[0025] S1, crushed and homogenized sea cucumber samples;

[0026] S2, take the sample obtained in S1, add the internal standard working solution, mix well and place in a dark place, then add anhydrous sodium sulfate, acidify acetonitrile and extract; concentrate the extract to obtain a residue;

[0027] S3. Dissolve the residue with acetonitrile aqueous solution, add n-hexane to remove fat, and take the clear liquid as a test sample.

[0028] The internal standard working solution involved in the present invention is added according to the standard and can be adjusted according to conventional techniques and requirements in the field.

[0029] In some embodiments, the internal standard working solution is a mixed solution of ciprofloxacin-D8, norfloxacin-D5, and enrofloxacin-D6, each with a concentration of 1.0 μg / mL.

[0030] In some embodiments, the mass ratio of the sample to anhydrous sodium sulfate is 1:2.

[0031] In some embodiments, the acetonitrile aqueous solution is a 5%-20% acetonitrile aqueous solution, preferably a 15% acetonitrile aqueous solution.

[0032] Beneficial Effects

[0033] In the environment where testing agencies use low-resolution liquid chromatography-tandem triple quadrupole mass spectrometry for rough screening of drug residues, more and more false positive cases are reported. This study provides a solution to the false positive problem of pefloxacin in complex matrices. Low-resolution triple quadrupole mass spectrometry is used to select m / z 219 at a specific energy as the quantitative ion; or the abundance ratio of the third product ion and its corresponding second daughter ion is selected to increase selectivity; quinolone drugs can be detected more efficiently and accurately.

[0034] Terminology

[0035] Certain embodiments of the present invention are now described in detail. The present invention is intended to cover all substitutions, modifications and equivalent technical solutions, which are all included in the scope of the present invention as defined in the claims. It should be appreciated by those skilled in the art that many methods and materials similar or equivalent to those described herein can be used to practice the present invention. The present invention is by no means limited to the methods and materials described herein. In the event that one or more of the combined documents, patents and similar materials are different from or contradictory to the present application (including but not limited to defined terms, term applications, described technologies, etc.), the present application shall prevail.

[0036] It should be further appreciated that certain features of the invention, which for clarity are described in multiple separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the invention, which for brevity are described in a single embodiment, may also be provided separately or in any suitable sub-combination.

[0037] Unless otherwise specified, all technical terms used in the present invention have the same meaning as commonly understood by those skilled in the art to which the present invention belongs. All patents and publications related to the present invention are incorporated herein by reference in their entirety.

[0038] In the description of this specification, the description with reference to the terms "one embodiment", "some embodiments", "example", "specific example", or "some examples" etc. means that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described may be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art may combine and combine the different embodiments or examples described in this specification and the features of the different embodiments or examples, without contradiction.

[0039] In the following, all numbers disclosed herein are approximate, regardless of whether the words "about" or "approximately" are used. The value of each number may vary by 1%, 2%, 5%, 7%, 8%, 10%, 15% or 20%. Whenever a number having a value of N is disclosed, any number having a value of N+ / -1%, N+ / -2%, N+ / -3%, N+ / -5%, N+ / -7%, N+ / -8%, N+ / -10%, N+ / -15% or N+ / -20% is explicitly disclosed, where "+ / -" means plus or minus. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1 Chromatograms and mass spectra of pefloxacin standard (a) and sample (b).

[0041] Figure 2 Extracted ion chromatograms of pefloxacin standard (a), sample (b), and sample spike (c) with changing chromatographic conditions.

[0042] Figure 3 MS of 1.0 μg / mL ciprofloxacin standard (a) and sample (b) 1 Spectra and MS of ciprofloxacin standard (c) and sample (d) 2 Spectrum diagram (NCE is 40).

[0043] Figure 4 Chromatograms and mass spectra of ciprofloxacin isotopes (a) and pefloxacin interfering peaks in the sample (b).

[0044] Figure 5 Mass fragmentation pathways of ciprofloxacin.

[0045] Figure 6 Mass fragmentation pathway of pefloxacin.

[0046] Figure 7 Variation trend of different ion intensities of ciprofloxacin (a) and its isotopes (b) and pefloxacin (c) with collision energy.

[0047] Figure 8 Chromatograms of 10 ng / mL pefloxacin (a), 1.0 μg / mL ciprofloxacin isotope (b), and sample (c).

[0048] Fig. 9 Mass spectra of pefloxacin standard (a), ciprofloxacin isotopes (b), and samples (c). DETAILED DESCRIPTION

[0049] In order to make the purpose, technical scheme and advantages of the present invention clearer, the present invention is further described in detail below in conjunction with embodiments. The specific embodiments described herein are only used to explain the present invention and are not intended to constitute any limitation of the present invention. In addition, in the following description, the description of known structures and technologies is omitted to avoid unnecessary confusion of the concepts of the present disclosure. Such structures and technologies are also described in many publications.

[0050] The reagents used in the present invention can be purchased from the market or prepared by the method described in the present invention.

[0051] 1. Chemical reagents:

[0052] Ciprofloxacin, enrofloxacin, pefloxacin, deuterated ciprofloxacin, deuterated norfloxacin, deuterated enrofloxacin standard (DR. Ehrenstorfer GmbH, Germany, purity ≥ 95.0%), methanol and acetonitrile were all chromatographically pure (Tedia, USA), the experimental water was ultrapure water prepared by Milli-Q system (Milli-Q ultrapure water system), and the other reagents were all analytically pure.

[0053] 2. Instruments and testing conditions:

[0054] TSQ Quantum ultra high performance liquid chromatography tandem triple quadrupole mass spectrometer and Q Exactive high performance liquid chromatography quadrupole-electrostatic field orbital trap high resolution mass spectrometer (Thermo Fisher, USA) were used for sample detection.

[0055] 3. Sample collection and pretreatment:

[0056] Sea cucumber samples were collected from the sea cucumber breeding base in Xiapu County, Ningde City, Fujian Province, China.

[0057] S1: Chop the sample, mix it and homogenize it for later use.

[0058] S2: Accurately weigh 5.0g of sample, add 50μL of mixed internal standard working solution (ciprofloxacin-D8, norfloxacin-D5, enrofloxacin-D6 mixed solution, each concentration is 1.0μg / mL) to a 50mL stoppered centrifuge tube, vortex mix for 30s, and place in the dark for 10min. Add 10g of anhydrous sodium sulfate, vortex mix, then add 20mL of acidified acetonitrile [acetonitrile + formic acid = 99 + 1 (v / v)], vortex mix for 1min, and ultrasonically extract for 10min. Centrifuge at 4000r / min for 5min, and take the supernatant into a 50mL round-bottom flask.

[0059] S3: Add 20 mL of acidified acetonitrile to the residue, repeat the extraction once, combine the two extracts, and rotary evaporate to dryness in a 40°C water bath to obtain the residue.

[0060] S4: Add 1 mL of 15% acetonitrile solution and vortex to dissolve the residue, then add 2 mL of n-hexane and vortex to mix for 30 seconds, transfer to a 5 mL stoppered centrifuge tube, centrifuge at 4000 r / min for 5 minutes, discard the upper layer, take the lower clear liquid, filter it through a 0.25 μm filter membrane, and prepare for detection by the machine.

[0061] 4. Detection and analysis:

[0062] 1. Detect samples using high performance liquid chromatography-tandem triple quadrupole mass spectrometry

[0063] This experiment adopted the commonly used method in the field for detection, that is, high performance liquid chromatography-tandem triple quadrupole mass spectrometry was used to detect and analyze the sea cucumber samples pretreated in this article.

[0064] HPLC conditions:

[0065] Chromatographic column: CAPCELL PAK C18 MGⅡ chromatographic column (2.1mm×150mm, 5μm); column temperature 30℃; flow rate 0.25mL / min; injection volume 10μL; mobile phase A: 0.1% (V / V) formic acid (5mM ammonium acetate)-water solution, mobile phase B: 0.1% formic acid acetonitrile. Gradient elution, elution program: 0~3.0min, 15%B~40%B; 3.0~9.0min, 40%B; 9.0~10.0min, 40%B~15%B; 10.0~15.0min, 15%B.

[0066] TSQ Quantum ultra high performance liquid phase tandem triple quadrupole mass spectrometer mass spectrometry conditions:

[0067] Positive ion scan mode;

[0068] Ionization mode: electrospray ESI ion source positive mode;

[0069] Spray voltage: 4000V;

[0070] Sheath gas flow: 35.0arb;

[0071] Auxiliary gas: 10.0arb;

[0072] Capillary temperature: 350℃;

[0073] Collision gas: argon;

[0074] Scanning mode: multiple reaction monitoring (SRM) mode.

[0075] The secondary fragment ion with the highest abundance was used as the quantitative ion, and the second most abundant fragment ion was used as the qualitative ion. The mass spectrometry parameters are shown in Table 1.

[0076] Table 1 Mass parameters of target compounds in HPLC-triple quadrupole mass spectrometry

[0077]

[0078] Note: *Specifies the amount of ions.

[0079] Analysis step 1: The retention time of the chromatographic peak of the pefloxacin standard substance is 4.30min ( Figure 1 , a), while the retention time of the chromatographic peak in the sample is 4.31min( Figure 1, b), the retention time difference is 0.01min, and the deviation is 0.233%, which meets the requirement that the retention time deviation is less than ±5%.

[0080] Analysis step 2: As shown in Table 1, pefloxacin was in positive ion scanning mode, with acetonitrile as the mobile phase, the parent ion was m / z 334, the daughter ion m / z 316 was the quantitative ion, and the daughter ion m / z 290 was the qualitative ion. By comparing the peak areas of the extracted secondary daughter ions, it was found that the abundance ratio of the daughter ions m / z 290 and m / z 316 of the pefloxacin standard substance (qualifier ion / quantitative ion×100%) was 51%, and the abundance ratio of this daughter ion pair in the chromatographic peak in the sample was 47%, and the relative deviation from the abundance ratio of the ion of the pefloxacin standard substance was -7.84%, which met the requirement of a relative deviation of less than ±20% according to the standard.

[0081] Based on the combined analysis of step 1 and step 2, the sample can be determined to be pefloxacin positive.

[0082] Comprehensive analysis: The actual detection rate of pefloxacin in aquatic products is low. A positive result appeared this time. In order to ensure the accuracy of the test results, other methods were used to confirm the samples. Pefloxacin standard was added to the sample, and the proportion of acetonitrile in the organic phase in the mobile phase was adjusted from 40% to 20%. It was found that the retention time of the chromatographic peak in the sample decreased with the decrease of the acetonitrile ratio. The difference between the elution time of the pefloxacin standard and the sample gradually increased. When the acetonitrile ratio was 20% and the gradient elution time was extended from 2.5min to 8min, the retention time deviation increased from 0.01min to 0.09min, indicating the possibility of false positives. For further confirmation, methanol with a slightly weaker elution force was replaced as mobile phase B, and it was found that the interference peak and the pefloxacin standard peak could be completely separated ( Figure 2 , a is the standard, b is the sample, and c is the sample with the standard added), thus confirming that the chromatographic peak in the sample is the interference peak of pefloxacin. Therefore, it is judged that pefloxacin in the sample is a false positive.

[0083] Conclusion: False positives of pefloxacin occurred in the detection of aquatic products by conventional methods, namely high performance liquid chromatography-tandem triple quadrupole mass spectrometry. If it is not confirmed by other methods, it is difficult to judge whether it is a false positive in unknown circumstances. The current high performance liquid chromatography-tandem triple quadrupole mass spectrometry method for the detection of quinolones has problems in its direct applicability.

[0084] 2. Identification of interferences by high performance liquid quadrupole-electrostatic field orbital trap high resolution mass spectrometry

[0085] In this experiment, Q Exactive high performance liquid chromatography quadrupole-electrostatic field orbital trap high resolution mass spectrometry was used to detect and analyze the interfering components of pefloxacin in the pretreated sea cucumber samples.

[0086] HPLC conditions:

[0087] Chromatographic column: CAPCELL PAK C18 MGⅡ chromatographic column (2.1mm×150mm, 5μm); column temperature 30℃; flow rate 0.25mL / min; injection volume 10μL; mobile phase: A: 0.1% (V / V) formic acid (5mM ammonium acetate)-water solution; B: methanol. Gradient elution, elution program: 0~3.0min, 15%~40%B; 3.0~9.0min, 40%B; 9.0~10.0min, 40%B~15%B; 10.0~15.0min, 15%B.

[0088] Q Exactive high performance liquid phase quadrupole-electrostatic field orbital trap high resolution mass spectrometer mass spectrometry conditions:

[0089] Positive ion scan mode,

[0090] Ionization mode: electrospray ESI ion source positive mode;

[0091] Spray voltage: 4000V;

[0092] Sheath gas flow: 35.0arb;

[0093] Auxiliary gas: 10.0arb;

[0094] Capillary temperature: 350℃;

[0095] Primary mass spectrometry full scan mode: resolution R = 70000; scanning range m / z 150-380;

[0096] Secondary mass spectrometry scanning mode: parallel reaction monitoring PRM: resolution R = 17500;

[0097] Normalized collision energy (NCE) 20, 30, 40, AGC target 2e 4 ;

[0098] Isolation window: m / z 2.0.

[0099] Analysis step 1: Use a liquid phase analysis method in which the mobile phase is changed to methanol to achieve chromatographic separation of the interference peak and pefloxacin.

[0100] Analysis step 2: Use Q Exactive high-resolution mass spectrometer to perform primary and secondary scans of the chromatographic peaks in the sample (see Figure 3 b, d). The spectra showed that only ciprofloxacin was detected, and the primary and secondary mass spectra of the sample and ciprofloxacin standard (where the target parent ion was 334.1561, and the isolation window was set to 0.8 m / z) were completely consistent (see Figure 3 a, c).

[0101] Analysis Step 3:

[0102] First, adjust the chromatographic separation method such as gradient and temperature to confirm whether the unknown substance and ciprofloxacin can be separated. If confirmed, proceed to the next step.

[0103] Secondly, when a single standard of ciprofloxacin was injected, in the positive ion scanning mode, an isotope peak of m / z 334 appeared in the primary spectrum, and two daughter ions of m / z 290 and m / z 316 appeared in the secondary spectrum. The chromatograms of ion extraction of the primary parent ion and the secondary daughter ion confirmed that the interfering peaks in the sample came from the ciprofloxacin isotopes.

[0104] Again, by comparing the peak areas of the extracted secondary ions, the sample ion abundance ratio was about 25%, and the ion abundance ratio of the standard ciprofloxacin isotope extracted ion chromatogram peak m / z 316 and m / z 290 was 21.09%. It can be seen that the ciprofloxacin isotope and the interference product ion abundance ratio in the sample are consistent, and the interference is judged to be the ciprofloxacin isotope peak (see Figure 4 ).

[0105] From the above analysis, it can be seen that if ciprofloxacin is present in an unknown product, it is easy to get a false positive result of pefloxacin if only one test is performed using conventional methods. Changing the chromatographic conditions for chromatographic separation can solve this problem to a certain extent, but this exploration process is very cumbersome for conventional high-pressure binary pumps. Therefore, it is necessary to find a more accurate, simple and low-cost way to detect whether pefloxacin exists in aquatic products.

[0106] The present invention compares and studies the daughter ions of pefloxacin and ciprofloxacin isotopes, and finds a selective, simple and low-cost detection method, which can quickly and accurately test whether it is pefloxacin.

[0107] Screening of characteristic product ions of pefloxacin:

[0108] In aquatic products, ciprofloxacin and enrofloxacin are restricted drugs, so the detection rate is relatively high. When ciprofloxacin is present in the sample, it is ineffective to identify pefloxacin false positives by changing the ciprofloxacin daughter ion pairs.

[0109] The analysis of the fragmentation patterns of ciprofloxacin and pefloxacin (Tang QF, Chen FL, Xin XF. Study of the fragmentation patterns of nine fluoroquinolones by tandem mass spectrometry [J]. Anal Lett, 2012, 45 (1): 43-50 and Ma B, Sun YM, Chen XY, et al. Analysis of Fluroquinolones by Electrospray Ion Trap Mass Spectrometry [J]. Journal of Chinese Mass Spectrometry Society, 2006, 27 (3): 163-167.) shows that under the (+) ESI conditions, these compounds are prone to produce [M+H] + ion.

[0110] Ciprofloxacin cleavage (eg Figure 5 ):

[0111] (1) Under induced dissociation conditions, the C-3 carboxyl group of ciprofloxacin can dissociate along two pathways: one is the removal of a water molecule to generate an ion fragment of 314.1299, and then after losing a molecule of cyclopropene, the main fragment ion peak is obtained by two rearrangements of the four-membered ring hydrogen, and then a molecule of CO is removed to form a fragment ion of m / z 203.0613; the other is the removal of a CO2 molecule to generate an ion fragment of m / z 288.1507, and then dissociate along two pathways: one is the cleavage of F at C-6 to remove a molecule of HF, which is manifested as the generation of a fragment ion with an accurate mass number of m / z 268.1438; the other is the cleavage of the piperazine ring at C-7 to remove a neutral molecule (CH2=CH-NH2) to obtain m / z 245.1084 fragment ion, m / z 245.1084 loses another molecule of cyclopropene to produce a fragment ion m / z 205.0775.

[0112] (2) The parent ion of ciprofloxacin can also lose a molecule of HF to generate m / z 312.1348; while m / z 204.0691 is a radical ion, which is generated by [M+H]+ losing a molecule of cyclopropene and then undergoing two four-membered ring hydrogen rearrangements (losing C2H5N) to obtain m / z 249.0673, and then losing the HCOO· radical at the 3-position C of m / z 249.0673 to generate the fragment ion m / z 204.0691. [M+H]+ can also first lose a molecule of HCOOH, and then undergo a four-membered ring hydrogen rearrangement and an α-breakage on the piperazine ring to produce the fragment ion m / z 217.0770.

[0113] Pefloxacin cleavage (eg Figure 6 ):

[0114] Pefloxacin [M+H]+m / z 334.1553. In the MS / MS spectrum of the [M+H]+ ion, an ion of m / z 316.1451 produced by dehydration of the C-3 carboxyl group can be observed, and then a fragment ion of m / z 288.1498 is generated after the loss of a molecule of CO. The ion m / z 290.1655 is generated by the loss of the carboxyl group, and its C-7 position is replaced by a piperazine ring. After decarboxylation, the piperazine ring part continues to lose 57Da (C3H7N) and 71Da (C4H9N) neutral molecules and free radicals to produce corresponding daughter ions m / z 233.1078 and m / z 219.0924.

[0115] The main fragment ions of ciprofloxacin and pefloxacin are summarized in Table 2. It can be seen from the table that, except for m / z 288 which is the same daughter ion, the other daughter ions of pefloxacin are exactly the same as the daughter ions of ciprofloxacin isotope (m / z 334). Although m / z 288 can be selected as the daughter ion to exclude the interference of ciprofloxacin isotope, ciprofloxacin has a crosstalk effect, which is easy to cause false positive detection of pefloxacin. Therefore, it is impossible to avoid the interference of ciprofloxacin by selecting specific daughter ions. When the present invention adopts the commonly used low-resolution triple quadrupole mass spectrometer for detection, by adjusting the collision energy and the collision gas pressure, the ion intensity difference between ciprofloxacin isotope and pefloxacin at the same collision energy is large, and preferably the difference reaches the difference between "with and without". Therefore, the variation trend of ion intensity with collision energy was investigated under different collision gas pressures. For example, when m / z 219 was used as a quantitative ion and the collision gas pressure was 1.0 mtorr, the interference with pefloxacin could be avoided when the collision energy was less than 12 eV ( Figure 7 ). Figure 8 It can be seen that changing the quantitative ion to m / z 219 and the collision energy to 8ev successfully screened out the interference of pefloxacin in the sample.

[0116] Table 2 MS / MS analysis of compounds

[0117]

[0118] By comparing the peak areas of the extracted secondary daughter ions, it was found that although the abundance ratio of m / z 290 and m / z 316 of the daughter ions of pefloxacin, ciprofloxacin and samples was about 45%, compared with the third daughter ion m / z 233, the abundance ratio of m / z 233 and m / z 290 in the pefloxacin standard was 22.8%, and the abundance ratio of m / z 233 and m / z 290 in the ciprofloxacin isotope and samples was 78.0% and 65.2%, respectively. This shows that false positive substances can be screened out by adding the third daughter ion and its abundance ratio with the second daughter ion. On the other hand, it also confirms that the false positive samples come from ciprofloxacin isotopes (such as Fig. 9 ).

[0119] In summary, this paper uses high-resolution mass spectrometry to analyze false positive samples of pefloxacin in sea cucumbers, and uses primary and secondary mass spectrometry for structural identification to determine that the interfering substance is a ciprofloxacin isotope. In the environment where testing agencies use a large number of low-resolution triple quadrupole mass spectrometers for rough screening of drug residues, more and more false positive cases are reported. The present invention provides a solution to the problem of false positive results of pefloxacin in the detection of quinolone drugs in complex matrices, avoiding false positive results of pefloxacin. The present invention provides a solution to the improvement of quinolone drug detection methods and the realization of accurate detection of quinolone drugs in complex matrices, which is of great significance, and also provides a reference method for the exclusion of other false positive drugs.

[0120] The method of the present invention has been described through preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the methods and applications described herein within the content, spirit and scope of the present invention to implement and apply the technology of the present invention. Those skilled in the art can refer to the content of this article and appropriately improve the process parameters. It is particularly important to point out that all similar replacements and modifications are obvious to those skilled in the art, and they are all considered to be included in the present invention.

Claims

1. A method for detecting quinolone drugs to eliminate false positives of pefloxacin, characterized in that: Liquid chromatography-low resolution triple quadrupole mass spectrometry was used to detect sea cucumber samples, and m / z 219 was used as the quantitative ion to confirm whether the samples contained pefloxacin.

2. The detection method according to claim 1, characterized in that: The abundance ratio of m / z 290 and m / z 316 was approximately 40%-50%, which further confirmed whether the sample contained pefloxacin.

3. The detection method according to claim 1 or 2, characterized in that: The abundance ratio of m / z 233 and m / z 290 was 20%-30%, which further confirmed whether the sample contained pefloxacin.

4. The detection method according to claim 1, characterized in that: The detection conditions of the liquid chromatography are: Chromatographic column: C18 column, 2.1mm×150mm, 5μm; Flow rate 0.2-0.3mL / min; Gradient elution: Mobile phase A: 0.1% (V / V) formic acid-water solution, 5 mM buffer salt; Mobile phase B: methanol or acetonitrile; Elution program: 0-3.0 min, 15%-40% mobile phase B; 3.0-9.0 min, 40% mobile phase B; 9.0-10.0 min, 40%-15% mobile phase B; 10.0~15.0min, 15% mobile phase B.

5. The detection method according to claim 4, characterized in that: Chromatographic column: CAPCELL PAK C18 MGⅡ chromatographic column, 2.1mm×150mm, 5μm; Flow rate: 0.25 mL / min; Gradient elution: mobile phase A: 0.1% (V / V) formic acid-water solution, 5 mM ammonium acetate; mobile phase B: acetonitrile; The elution program was: 0-3.0 min, 15%-40% mobile phase B; 3.0-9.0 min, 40% mobile phase B; 9.0-10.0 min, 40%-15% mobile phase B; 10.0~15.0min, 15% mobile phase B.

6. The detection method according to claim 1, characterized in that: The low-resolution triple quadrupole mass spectrometry detection conditions were as follows: Ionization mode: electrospray ESI ion source positive mode; Spray voltage: 4000V; Sheath gas flow: 35.0arb; Auxiliary gas: 10.0arb; Capillary temperature: 350℃; Collision gas: argon; Scanning mode: multiple reaction monitoring (SRM) mode.

7. The detection method according to claim 1, characterized in that: The sea cucumber sample is pretreated: S1, crushed and homogenized sea cucumber samples; S2, taking the sea cucumber sample obtained in S1, adding the internal standard working solution, mixing and placing in a dark place, adding anhydrous sodium sulfate, acidifying acetonitrile, and extracting; concentrating the extract to obtain a residue; S3. Dissolve the residue with acetonitrile aqueous solution, add n-hexane to remove fat, and take the clear liquid as a test sample.

8. The detection method according to claim 7, characterized in that: The internal standard working solution is a mixed solution of ciprofloxacin-D8, norfloxacin-D5, and enrofloxacin-D6, and the concentration of each solution is 1.0 μg / mL.

9. The detection method according to claim 7, characterized in that: The mass ratio of the sea cucumber sample to anhydrous sodium sulfate is 1:

2.

10. The detection method according to claim 7, characterized in that: The acetonitrile aqueous solution is a 5% to 20% acetonitrile aqueous solution.

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