CN111665301A - Kit for detecting antifungal drugs in serum by ultra-high performance liquid chromatography tandem mass spectrometry technology - Google Patents
Kit for detecting antifungal drugs in serum by ultra-high performance liquid chromatography tandem mass spectrometry technology Download PDFInfo
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- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
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Abstract
The invention relates to a kit for detecting antifungal drugs in serum by using an ultra-high performance liquid chromatography tandem mass spectrometry technology, which has the advantages of simple pretreatment process, low cost, high sensitivity and strong specificity, completes the separation and detection of antifungal drugs within 5min, basically meets the requirements on accuracy and precision, can be used for quantitative analysis of clinical antifungal drugs, and provides a reliable detection method for monitoring the treatment concentration of clinical antifungal drugs.
Description
Technical Field
The invention belongs to the technical field of serum detection, and particularly relates to a kit for detecting antifungal drugs in serum by using an ultra-high performance liquid chromatography tandem mass spectrometry technology, wherein the specific drugs are as follows: fluconazole (FCZ), Voriconazole (VCZ), Voriconazole N-Oxide (OVCZ), Posaconazole (PCZ), Itraconazole (Itraconazole, ICZ), Hydroxyitraconazole (HICZ), Caspofungin (CPF).
Background
Fungal infections are a clinically considerable problem, with fungal infections accounting for about 7.5% and even higher in nosocomial infections. Especially for patients with critical illness, organ transplantation, immunodeficiency, and immunosuppressive therapy. Fungal infections cause these patients to have a very high rate of infection and mortality, and there are few antifungal drugs available that are approved for clinical use, and some of these drugs are resistant, making treatment of antifungal infections more difficult, and therefore more rational use of existing drugs is needed. Fluconazole (FCZ), Voriconazole (VCZ), Posaconazole (PCZ), Itraconazole (Itraconazole, ICZ), Caspofungin (CPF) are commonly used antifungal drugs. Wherein, the fluconazole, the voriconazole, the posaconazole and the itraconazole belong to triazole antifungal drugs. Fluconazole is a broad-spectrum antifungal drug, but has a narrow therapeutic window and high blood concentration with strong toxicity. Voriconazole belongs to a new generation of broad spectrum triazole antifungal drugs, has an antibacterial spectrum and antibacterial activity greater than that of fluconazole, and is an antifungal drug widely applied clinically at present. The voriconazole has large drug metabolism individual difference in a patient body, complex pharmacokinetics and can interact with various drugs such as cyclosporin A and the like, so that the blood concentration and clinical treatment effect of the voriconazole are poor, even adverse reaction and toxicity are caused, and the metabolite voriconazole nitrogen oxide also has weak antibacterial activity, so that the understanding of the concentration of the metabolite in the body is beneficial to determining the effect of the voriconazole and the metabolite thereof on the organism, so that the drug treatment is more effective and safer. Posaconazole is a high-efficiency, low-toxicity and broad-spectrum medicine for treating and preventing invasive fungal infection, has a good curative effect on fungi with drug resistance to polyenes and other triazole antifungal medicines, is easy to generate drug interaction when being combined with other medicines because the posaconazole can generate interaction with CYP3A4, and is very necessary to monitor the drug concentration in clinical use. Itraconazole, which is also a triazole antifungal drug, is used to treat severe or refractory skin fungal infections by inhibiting yeast and major components of fungal cell membranes, and is metabolized in the liver into various metabolites, of which Hydroxyitraconazole (HICZ) is a major metabolite, and has similar antibacterial effects to itraconazole. However, itraconazole has poor water solubility and poor absorption after oral administration, thereby resulting in low effective blood concentration in vivo and large individual difference. Itraconazole interacts with various drugs, so that it is required to monitor plasma concentration of itraconazole and its metabolites, hydroxyitraconazole, in vivo at the time of clinical use. Caspofungin is a first-line antifungal drug, belongs to echinocandin drugs, has good antifungal effect, is safe to human bodies, is often used for treating and preventing drug resistance of azole drugs or candida infection of critically ill patients, and needs to monitor the drug concentration and the drug absorption specificity of the critically ill patients.
At present, methods for detecting the concentration of antifungal drugs in an object mainly include a microbiological method, a high performance liquid chromatography and an ultra-high performance liquid-tandem mass spectrometry, most of literature reports are used for detecting 1-4 drugs at one time, and patent reports are also provided, such as Chinese patent application (publication number: CN 109030672A) which discloses one of the following methods: the kit for simultaneously determining the four antifungal drugs in the dry blood spots and the application thereof aim at detecting the 4 antifungal drugs, and a single sample has long acquisition time and is not suitable for a linear range; the chinese patent application (publication No. CN 110470775 a) discloses: the kit and the detection method thereof for monitoring the concentration of fluconazole in blood only aim at one drug, special equipment needs to be configured, the applicability is not strong, the sample size requirement is large, the sample pretreatment is complex, and the defects of matrix effect interference and the like exist. The antifungal medicine provided by the invention adopts an LC-MS/MS method, can detect 7 common antifungal medicines at one time, has high sensitivity and good accuracy, can be well applied to clinic, and can provide a reliable basis for antifungal medicine combination research.
Disclosure of Invention
The invention aims to provide a kit for detecting antifungal drugs in serum by using an ultra-high performance liquid chromatography tandem mass spectrometry technology on the basis of the prior art.
The invention also aims to provide application of the kit in detection of antifungal drugs in serum by using an ultra-high performance liquid chromatography tandem mass spectrometry technology.
The technical scheme of the invention is as follows:
a reagent kit for detecting antifungal drugs in serum by an ultra-high performance liquid chromatography tandem mass spectrometry technology,
the antifungal drugs are respectively: fluconazole (FCZ), Voriconazole (VCZ), voriconazole N-Oxide (OVCZ), Posaconazole (PCZ), Itraconazole (Itraconazole, ICZ), Hydroxyitraconazole (HICZ), Caspofungin (CPF)
The isotope internal standard substances corresponding to the antifungal drugs are respectively as follows: voriconazole-d 3(VCZ-d3), voriconazole N-oxide-d 3(OVCZ-d3), fluconazole-d 4(FCZ-d4), itraconazole-d 5(ICZ-d5), hydroxyitraconazole-d 5(HICZ-d5), posaconazole-d 4(PCZ-d4), caspofungin-d 4(CPF-d 4).
The kit comprises the following reagents:
(1) mobile phase:
mobile phase A: 0.01 to 0.2 percent of formic acid aqueous solution; mobile phase B: acetonitrile;
(2) a calibration sample:
calibrator samples at seven different concentration points S7-S1:
s7: contains CPF 20000ng/mL, VCZ/OVCZ/ICZ/HICZ 10000ng/mL, FCZ 40000ng/mL, PCZ5000 ng/mL;
s6: contains CPF10000ng/mL, VCZ/OVCZ/ICZ/HICZ 5000, FCZ 20000ng/mL, PCZ 2500 ng/mL;
s5: contains CPF 5000ng/mL, VCZ/OVCZ/ICZ/HICZ 2500, FCZ 10000ng/mL, PCZ 1250 ng/mL;
s4: contains CPF1000 ng/mL, VCZ/OVCZ/ICZ/HICZ 500, FCZ 2000ng/mL, PCZ 250 ng/mL;
s3: contains CPF 500ng/mL, VCZ/OVCZ/ICZ/HICZ 250, FCZ 1000ng/mL, PCZ 125 ng/mL;
s2: contains 200ng/mL CPF, 200ng/mL VCZ/OVCZ/ICZ/HICZ 100, 400ng/mL FCZ and 50ng/mL PCZ;
s1: contains CPF100 ng/mL, VCZ/OVCZ/ICZ/HICZ 50, FCZ 200ng/mL, PCZ 25 ng/mL;
(3) mixing internal standard solutions:
comprises methanol water solution of FCZ-d44000ng/mL, VCZ-d31000ng/mL, CPF-d42000ng/mL, OVCZ-d31000ng/mL, PCZ-d 42000ng/mL, ICZ-d 55000 ng/mL and HICZ-d 54000 ng/mL;
(4) protein precipitant:
a mixed solution of methanol and acetonitrile;
(5) quality control product:
the serum matrix solution containing antifungal medicine has low, medium and high concentrations of QC (L), QC (M) and QC (H), respectively,
QC (L) includes: FCZ 400ng/mL, VCZ 100ng/mL, CPF 200ng/mL, OVCZ 100ng/mL, PCZ50ng/mL, ICZ 100ng/mL, HICZ 100 ng/mL;
QC (M) comprises: FCZ 4000ng/mL, VCZ 1000ng/mL, CPF 2000ng/mL, OVCZ 1000ng/mL, PCZ500ng/mL, ICZ 1000ng/mL, HICZ 1000 ng/mL;
QC (H) includes: FCZ 16000ng/mL, VCZ 4000ng/mL, CPF 8000ng/mL, OVCZ 4000ng/mL, PCZ2000ng/mL, ICZ 4000ng/mL, HICZ 4000 ng/mL.
A reagent kit for detecting antifungal drugs in serum by an ultra-high performance liquid chromatography tandem mass spectrometry technology,
the antifungal drugs are respectively: FCZ, VCZ, OVCZ, PCZ, ICZ, HICZ, CPF.
The kit comprises the following reagents:
(1) eluent:
mobile phase A: 0.01 to 0.2 percent of formic acid aqueous solution, and a mobile phase B: acetonitrile;
(2) a calibration sample:
mixed standard solution containing FCZ 800000ng/mL, VCZ 200000ng/mL, CPF 400000ng/mL, OVCZ 200000ng/mL, PCZ100000ng/mL, ICZ200000ng/mL, HICZ200000ng/mL and blank serum matrix; before detection, calibration sample samples S7-S1 at seven different concentration points and quality control solution QC (L), QC (M) and QC (H) at three concentrations of low, medium and high are prepared by dilution according to the following formula recorded in an instruction book:
s7: contains CPF 20000ng/mL, VCZ/OVCZ/ICZ/HICZ 10000ng/mL, FCZ 40000ng/mL, PCZ5000 ng/mL;
s6: contains CPF10000ng/mL, VCZ/OVCZ/ICZ/HICZ 5000, FCZ 20000ng/mL, PCZ 2500 ng/mL;
s5: contains CPF 5000ng/mL, VCZ/OVCZ/ICZ/HICZ 2500, FCZ 10000ng/mL, PCZ 1250 ng/mL;
s4: contains CPF1000 ng/mL, VCZ/OVCZ/ICZ/HICZ 500, FCZ 2000ng/mL, PCZ 250 ng/mL;
s3: contains CPF 500ng/mL, VCZ/OVCZ/ICZ/HICZ 250, FCZ 1000ng/mL, PCZ 125 ng/mL;
s2: contains 200ng/mL CPF, 200ng/mL VCZ/OVCZ/ICZ/HICZ 100, 400ng/mL FCZ and 50ng/mL PCZ;
s1: contains CPF100 ng/mL, VCZ/OVCZ/ICZ/HICZ 50, FCZ 200ng/mL, PCZ 25 ng/mL;
QC (L) includes: FCZ 400ng/mL, VCZ 100ng/mL, CPF 200ng/mL, OVCZ 100ng/mL, PCZ50ng/mL, ICZ 100ng/mL, HICZ 100 ng/mL;
QC (M) comprises: FCZ 4000ng/mL, VCZ 1000ng/mL, CPF 2000ng/mL, OVCZ 1000ng/mL, PCZ500ng/mL, ICZ 1000ng/mL, HICZ 1000 ng/mL;
QC (H) includes: FCZ 16000ng/mL, VCZ 4000ng/mL, CPF 8000ng/mL, OVCZ 4000ng/mL, PCZ2000ng/mL, ICZ 4000ng/mL, HICZ 4000 ng/mL;
(3) mixing internal standard solutions:
comprises methanol water solution of FCZ-d44000ng/mL, VCZ-d31000ng/mL, CPF-d42000ng/mL, OVCZ-d31000ng/mL, PCZ-d 42000ng/mL, ICZ-d 55000 ng/mL and HICZ-d 54000 ng/mL;
(4) protein precipitant:
a mixed solution of methanol and acetonitrile;
(5) the use instruction comprises: comprises calibrator samples S1-S7 and quality control solution QC (L), QC (M) and QC (H) with low, medium and high concentrations.
In a preferred embodiment, the mobile phase a is 0.01% to 0.2% aqueous formic acid, preferably 0.1% aqueous formic acid.
In one scheme, the volume ratio of methanol to acetonitrile in the protein precipitator is 1: 1-5; preferably, the volume ratio of methanol to acetonitrile in the protein precipitant is 1: 4.
The mixed standard solution mentioned in the present invention is prepared as follows:
the antifungal drugs FCZ 50mg/mL, VCZ 20mg/mL, CPF 5mg/mL, OVCZ 1mg/mL, PCZ1mg/mL, ICZ 2mg/mL, HICZ 0.5mg/mL are respectively prepared into mixed standard solutions containing FCZ 800000ng/mL, VCZ 200000ng/mL, CPF 400000ng/mL, OVCZ 200000ng/mL, PCZ100000ng/mL, ICZ200000ng/mL and HICZ 0 200000ng/mL by using methanol.
In a more preferred embodiment, the mixed standard solution is prepared as follows:
accurately transferring antifungal drug standard mother liquor with a certain volume respectively, adding 94 mu L of 80% methanol aqueous solution, and fully and uniformly mixing to obtain 1mL of mixed standard solution with the concentration shown in the following table 1.
TABLE 1 Mixed Standard solution
| Numbering | Components | Mother liquor concentration (μ g/mL) | Volume removal (mu L) | Total volume (μ L) | Concentration (ng/mL) |
| 1 | FCZ | 50 | 16 | 1000 | 800000 |
| 2 | VCZ | 20 | 10 | 1000 | 200000 |
| 3 | |
5 | 80 | 1000 | 400000 |
| 4 | OVCZ | 1 | 200 | 1000 | 200000 |
| 5 | PCZ | 1 | 100 | 1000 | 100000 |
| 6 | ICZ | 2 | 100 | 1000 | 200000 |
| 7 | HICZ | 0.5 | 400 | 1000 | 200000 |
Preparing standard yeast by gradient dilution method, taking out standard solution from refrigerator at-20 deg.C, vortex for 10s, preparing maximum concentration point of standard yeast with standard solution within 2min, and storing at-80 deg.C. The specific procedure is as follows in Table 2 (concentration unit: ng/mL):
TABLE 2 Standard koji preparation
| Calibration article | Pipetting solution (mu L) | Blank serum (mu L) | CPF | VCZ/OVCZ/ICZ/HICZ | FCZ | PCZ |
| S7 | Mixing of standard solution 10 | 190 | 20000 | 10000 | 40000 | 5000 |
| S6 | S7 50 | 50 | 10000 | 5000 | 20000 | 2500 |
| S5 | S7 40 | 120 | 5000 | 2500 | 10000 | 1250 |
| S4 | S6 20 | 180 | 1000 | 500 | 2000 | 250 |
| S3 | S5 20 | 180 | 500 | 250 | 1000 | 125 |
| S2 | S4 50 | 200 | 200 | 100 | 400 | 50 |
| S1 | S350 | 200 | 100 | 50 | 200 | 25 |
The mixed internal standard working solution provided by the invention is prepared according to the following method: FCZ-d 41 mg/mL, VCZ-d30.1mg/mL, CPF-d 40.5mg/mL, OVCZ-d31 mg/mL, PCZ-d40.5mg/mL, ICZ-d 50.5mg/mL, HICZ-d 51 mg/mL mother liquor is prepared into an isotope internal standard solution containing FCZ-d44000ng/mL, VCZ-d31000ng/mL, CPF-d42000ng/mL, OVCZ-d310 31000ng/mL, PCZ-d 42000ng/mL, ICZ-d 55000 ng/mL and HICZ-d 54000 ng/mL by using a methanol water solution.
In a more preferred embodiment, the mixed internal standard working solution is prepared according to the following method:
and accurately transferring a certain volume of antifungal drug isotope internal standard mother liquor, adding 963 mu L of 80% methanol aqueous solution, and uniformly mixing to obtain 1mL of isotope internal standard solution with the concentration shown in the following table 3.
TABLE 3 Mixed internal standard working solution formulation
When preparing a mixed standard solution and a mixed internal standard working solution, the adopted methanol aqueous solution is 50-95% methanol aqueous solution; preferably 70 to 90 percent of methanol aqueous solution; more preferably 80% aqueous methanol.
In the preparation of the mixed standard solution, the non-interfering serum matrix is blank serum without antifungal drugs.
The concentration of the aqueous methanol solution referred to in the present invention generally means a volume concentration.
The preparation method of the quality control solution QC (L), QC (M) and QC (H) with low, medium and high concentrations described in the specification is as follows:
QC (L) is QC (M) and is diluted to 10 times by using an interference-free serum substrate;
QC (M) is the mixed standard solution diluted to 200 times by using an interference-free serum substrate;
QC (H) is the above mixed standard solution diluted 50-fold with non-interfering serum base.
The serum mentioned in the invention is human or animal serum.
The application of the kit in detecting antifungal drugs in serum by using the ultra-performance liquid chromatography tandem mass spectrometry technology is also within the protection scope of the invention.
The specific detection method comprises the following steps:
a method for detecting antifungal drugs in serum by an ultra-high performance liquid chromatography tandem mass spectrometry technology,
the antifungal drugs are respectively: fluconazole (FCZ), Voriconazole (VCZ), voriconazole N-Oxide (OVCZ), Posaconazole (PCZ), Itraconazole (Itraconazole, ICZ), Hydroxyitraconazole (HICZ), Caspofungin (CPF)
The isotope internal standard substances corresponding to the antifungal drugs are respectively as follows: voriconazole-d 3(VCZ-d3), voriconazole N-oxide-d 3(OVCZ-d3), fluconazole-d 4(FCZ-d4), itraconazole-d 5(ICZ-d5), hydroxyitraconazole-d 5(HICZ-d5), posaconazole-d 4(PCZ-d4), caspofungin-d 4(CPF-d 4).
Detecting the antifungal drugs in the preprocessed serum by adopting an ultra-high performance liquid chromatography tandem mass spectrometry technology, firstly separating a target substance to be detected from interfering components in a serum matrix by utilizing the ultra-high performance liquid chromatography, then establishing a calibration curve by utilizing a mass spectrum isotope internal standard quantitative method and taking the concentration ratio of a standard substance to an internal standard substance as an X axis and the peak area ratio of the standard substance to the internal standard substance as a Y axis, and calculating the content of the antifungal drugs in the serum, wherein the specific chromatographic conditions are as follows:
(1) ultra-high performance liquid chromatography conditions:
mobile phase A: 0.01 to 0.2 percent of formic acid aqueous solution, preferably 0.1 percent of formic acid-aqueous solution; mobile phase B: acetonitrile;
the type of the chromatographic column: agilent Zorbax Eclipse XDB C18 (3X 100mm,1.8 μm);
the method adopts a gradient elution mode with the mobile phase A and the mobile phase B as a mixed mobile phase, the initial ratio of the mobile phase A to the mobile phase B is 95:5, and the volume ratio of the mobile phase A to the mobile phase B is gradually changed from 95:5 to 70:30 at a constant speed within 0-1.0 min; the volume ratio of the mobile phase A to the mobile phase B is gradually changed from 70:30 to 2:98 at a constant speed within 1.0-3.0 minutes; the volume ratio of the mobile phase A to the mobile phase B is changed from 2:98 to 95:5 within 3.0-5.0 minutes; in one embodiment, the flow rate of the mobile phase is 0.1-0.4 mL/min, preferably 0.3 mL/min. Further, the column temperature is 35-50 ℃, preferably 45 ℃. Furthermore, the injection volume is 1-5 μ L, preferably 1 μ L.
TABLE 4 mobile phase gradient elution parameters
(2) Mass spectrum conditions:
performing positive ion scanning in an electrospray ionization (ESI) mode by using Multiple Reaction Monitoring (MRM); the spray voltage was 3.0kV (ESI +); the source temperature is 120 ℃; the temperature of atomizing gas is 400 ℃, the airflow speed of atomizing is 800L/h, and the airflow speed of taper holes is 150L/h; each target and its corresponding isotope internal standard were monitored simultaneously.
The mass spectrometric acquisition parameters for each target analyte are shown in table 5.
TABLE 5 antifungal substance Spectrum parameters
| Compound (I) | Parent ion | Daughter ions | Declustering voltage (V) | Collision voltage (V) |
| FCZ | 307.1 | 127 | 14 | 28 |
| FCZ-d4 | 311.1 | 129 | 30 | 28 |
| VCZ | 350.1 | 154.8 | 28 | 24 |
| VCZ-d3 | 353.1 | 224.1 | 48 | 14 |
| OVCZ | 366.2 | 224.1 | 2 | 10 |
| OVCZ-d3 | 369.2 | 227.1 | 4 | 12 |
| CPF | 547.6 | 131 | 2 | 24 |
| CPF-d4 | 551.6 | 135 | 5 | 28 |
| PCZ | 701.3 | 127 | 20 | 48 |
| PCZ-d4 | 705.4 | 127.1 | 60 | 48 |
| ICZ | 705.3 | 392.3 | 76 | 32 |
| ICZ-d5 | 710.5 | 397.4 | 78 | 34 |
| HICZ | 721.4 | 408.3 | 68 | 34 |
| HICZ-d5 | 726.4 | 413.3 | 50 | 30 |
In order to improve the chromatographic separation selectivity, it may be considered to adjust the polarity of the mobile phase. The invention adds formic acid into the mobile phase A, can effectively improve the ionization efficiency of certain target compounds, has higher sensitivity for detecting antifungal drugs in serum by adopting an LC-MS/MS method in the prior art under the coordination of other conditions, has simple pretreatment process, low cost, high sensitivity and strong specificity, and completes the separation and detection of antifungal drugs within 5 min. In a preferable embodiment, the mobile phase a is 0.01% to 0.2% formic acid aqueous solution without affecting the effect of the present invention. In a more preferred embodiment, mobile phase a is 0.1% aqueous formic acid.
In chromatography, the choice of the chromatographic column is important and the requirements for the chromatographic column: high column efficiency, good selectivity, high analysis speed and the like. The invention adopts 0.01 to 0.2 percent formic acid water solution and acetonitrile as mobile phases, and the types of chromatographic columns are as follows: the AgilentZorbax Eclipse XDB C18(3 multiplied by 100mm,1.8 mu m), under the coordination of other conditions, the endogenous substance does not interfere the determination of the sample, the sensitivity is high, the specificity is strong, the cost is low, the pretreatment process is simple, the separation and the detection can be completed within 5.0min, and the precision and the accuracy can meet the requirements.
When the internal standard method is adopted, the selection of the internal standard substance is very important work. The ideal internal standard should be capable of being added to the sample in an accurate, known amount, and have substantially the same or as consistent as possible physicochemical properties, chromatographic behavior, and response characteristics as the sample being analyzed; under chromatographic conditions, the internal standard must be sufficiently separated from the components of the sample. The method adopts voriconazole-d 3(VCZ-d3), voriconazole N-oxide-d 3(OVCZ-d3), fluconazole-d 4(FCZ-d4), itraconazole-d 5(ICZ-d5), hydroxyitraconazole-d 5(HICZ-d5), posaconazole-d 4(PCZ-d4) and caspofungin-d 4(CPF-d4) as internal standards respectively, the deuterium internal standard and a substance to be tested have the same retention time, chemical properties and matrix effect, and the reproducibility and accuracy in the determination of the antifungal drug in serum are better.
The serum mentioned in the invention is human or animal serum.
In one protocol, pre-treated serum was prepared as follows: adding a protein precipitant containing an internal standard into the serum, and taking a supernatant after shaking and centrifuging; wherein the protein precipitator is a mixed solution of methanol and acetonitrile.
Preferably, the volume ratio of methanol to acetonitrile in the protein precipitant is 1: 1-5, without affecting the effect of the present invention, for example, the volume ratio of methanol to acetonitrile in the protein precipitant is 1: 4.
In a preferred embodiment, the pre-treated serum is prepared as follows: 50 mu L of plasma is taken and put into a 1.5mL centrifuge tube, 200 mu L of protein precipitator (the volume ratio of methanol to acetonitrile is 1: 1-5) containing internal standard is added into the centrifuge tube, and 60 mu L of supernatant is taken after centrifugation is carried out for 4-10 min at 12000-15000 r/min and 1-5 ℃. The protein precipitant containing internal standard is prepared by mixing mixed internal standard solution and protein precipitant, and the ratio of the internal standard to the protein precipitant is 0.1-0.3: 19.7-19.9.
In a more preferred embodiment, the pre-treated serum is prepared as follows: putting 50 μ L of plasma into a 1.5mL centrifuge tube, adding 200 μ L of protein precipitant containing internal standard (volume ratio of methanol to acetonitrile is 1:4), and oscillating at high speed (maximum oscillation speed) for 5 min; centrifuging at 14000r/min at 4 ℃ for 5 min; transfer 60. mu.L of supernatant from the EP tube to a plastic lined tube in a 1. mu.L sample volume. The protein precipitant containing the internal standard is prepared by mixing a mixed internal standard solution and the protein precipitant, wherein the ratio of the internal standard to the protein precipitant is 0.2:19.8, which is the same as the following.
Taking 50 mu L of each calibrator sample, putting the 50 mu L of each calibrator sample into a 1.5mL centrifuge tube, adding 200 mu L of protein precipitant containing an internal standard (the volume ratio of methanol to acetonitrile is 1:4), and oscillating at a high speed (maximum oscillation speed) for 5 min; centrifuging at 14000r/min at 4 ℃ for 5 min; transfer 60. mu.L of supernatant from the EP tube to a plastic lined tube in a 1. mu.L sample volume.
By adopting the technical scheme of the invention, the advantages are as follows:
when the kit provided by the invention is used for detecting the antifungal drugs in the serum, the pretreatment process is simple, the cost is low, the sensitivity is high, the specificity is strong, the separation and detection of the antifungal drugs are completed within 5min, the accuracy and the precision basically meet the requirements, the kit can be used for quantitative analysis of the antifungal drugs in clinic, and a reliable detection method is provided for monitoring the treatment concentration of the antifungal drugs in clinic.
Drawings
FIG. 1 is an extracted ion flow spectrum of an antifungal drug standard;
FIG. 2 is an ion flowgram of antifungal drug extracts in a serum sample.
Detailed Description
The invention will be better understood from the following examples. However, those skilled in the art will readily appreciate that the description of the embodiments is only for illustrating the present invention and should not be taken as limiting the invention as detailed in the claims.
Example 1:
first, experimental material and instrument
1. Material
The samples for the study experiments in the diagnostics were obtained from serum samples collected from the 9 month clinic of shanghai zhongshan hospital, 2019.
(1) The instrument comprises the following steps: xevo TQ-S triple quadrupole mass spectrometer (Waters Corporation); UPLC I-Class ultra high performance liquid chromatography system (with autosampler, Waters Corporation); SCILOGEX D2012 high speed bench top centrifuge (usa); ultra pure water meter (ELGA LabWater, uk); multi-tube Vortex mixer (Vortex genie2, usa); an adjustable pipettor (Eppendorf 0.5-10 muL, 10-100 muL, 100-1000 muL); glassware, graduated cylinders, and the like.
(2) Reagent consumables: MS grade methanol (Fisher, usa); MS grade acetonitrile (Fisher, usa); HPLC grade acetonitrile (Honeywell, usa); HPLC grade methanol (Honeywell, usa); MS grade formic acid (Fisher, usa); HPLC grade methanol (Honeywell, usa); chromatography column Agilent Zorbax Eclipse XDB C18 (3X 100mm,1.8 μm).
(3) And (3) standard substance: the standards and their corresponding internal standards are shown in table 6 below.
TABLE 6 Standard and internal standards
| Serial number | Name of Chinese | Manufacturer of the product |
| 1 | Voriconazole | TRC |
| 2 | Voriconazole-d 3 | TRC |
| 3 | Voriconazole N-oxide | TRC |
| 4 | Voriconazole N-oxide-d 3 | Shenzhen Zhenqiang Biotechnology Co.,Ltd. |
| 5 | Fluconazole | TRC |
| 6 | Fluconazole-d 4 | |
| 7 | Itraconazole | TRC |
| 8 | Itraconazole- |
TRC |
| 9 | Hydroxyitraconazole | TRC |
| 10 | Hydroxyitraconazole- |
TRC |
| 11 | Posaconazole | TRC |
| 12 | Posaconazole-d 4 | TRC |
| 13 | Caspofungin | TRC |
| 14 | Caspofungin-d 4 | TRC |
(4) Quality control product: the non-interference serum matrix containing antifungal medicine has low, medium and high concentration, which are QC (L), QC (M) and QC (H), as shown in Table 1.
The upper and lower peripheries of the kit are coated with membranes, shockproof and heat preservation are carried out, mobile phases A and B are placed at the upper left part, 11 ampoule bottles are respectively placed at the lower left part, and 7 calibrator samples, 1 mixed internal standard solution and quality control substances (QC (L), QC (M) and QC (H)) are respectively arranged; to the right, 25mL of protein precipitant was placed.
Second, liquid condition
(1) Chromatographic conditions are as follows: mobile phase A: 0.1% formic acid-water solution; mobile phase B: and (3) acetonitrile. The type of the chromatographic column: AgilentZorbax Eclipse XDB C18 (3X 100mm,1.8 μm) was eluted using a gradient as detailed in Table 5. The flow rate was 0.3mL/min, the column temperature was 45 ℃ and the injection volume was 1. mu.L.
(2) Mass spectrum conditions: in an electrospray ionization detection mode, adopting a mass spectrum scanning mode of multi-reaction monitoring; the spray voltage was 3.0kV (ESI +); the source temperature is 120 ℃; the temperature of atomizing gas is 400 ℃, the airflow speed of atomizing is 800L/h, and the airflow speed of taper holes is 150L/h; each target was monitored simultaneously with the isotope internal standard. The mass spectrometric acquisition parameters for each target analyte are shown in table 6.
Second, the experimental procedure
(1) Preparing a standard substance:
respectively mixing antifungal drug mother liquor with the following concentrations: FCZ 50mg/mL, VCZ 20mg/mL, CPF 5mg/mL, OVCZ 1mg/mL, PCZ1mg/mL, ICZ 2mg/mL, HICZ 0.5mg/mL were formulated with 80% aqueous methanol to comprise a mixed standard solution containing FCZ 800000ng/mL, VCZ 200000ng/mL, CPF 400000ng/mL, OVCZ 200000ng/mL, PCZ100000ng/mL, ICZ200000ng/mL, HICZ 0ng/mL (see Table 3 for details).
The mixed standard solution is prepared into calibration sample (see table 4) of seven different concentration points by using a blank serum matrix, the concentration of each calibration point is as listed in table 4, and the seven concentration points of the calibration sample are as follows:
CPF：100ng/mL、200ng/mL、500ng/mL、1000ng/mL、5000ng/mL、10000ng/mL、20000ng/mL
VCZ/OVCZ/ICZ/HICZ：50ng/mL、100ng/mL、250ng/mL、500ng/mL、2500ng/mL、5000ng/mL、10000ng/mL
FCZ：200ng/mL、400ng/mL、1000ng/mL、2000ng/mL、10000ng/mL、20000ng/mL、40000ng/mL
PCZ：25ng/mL、50ng/mL、125ng/mL、250ng/mL、1250ng/mL、2500ng/mL、5000ng/mL
(2) preparation of mixed internal standard solution
FCZ-d 41 mg/mL, VCZ-d30.1mg/mL, CPF-d 40.5mg/mL, OVCZ-d31 mg/mL, PCZ-d40.5mg/mL, ICZ-d 50.5mg/mL, HICZ-d 51 mg/mL mother liquor is prepared into isotope internal standard solutions containing FCZ-d44000 440 44000ng/mL, VCZ-d31000ng/mL, CPF-d42000ng/mL, OVCZ-d31000ng/mL, PCZ-d 42000ng/mL, ICZ-d 55000 ng/mL and HICZ-d 54000 ng/mL by using 80% methanol aqueous solution (see Table 2 for details);
(3) preparing a quality control product:
the mixed standard solution is prepared into QC (L), QC (M) and QC (H) with three different concentrations by using blank serum without antifungal drugs, and the details are shown in a table 7.
TABLE 7 quality control of antituberculosis drugs corresponding concentration (unit: ng/mL)
| Serial number | Compound (I) | QC(L) | QC(M) | QC(H) |
| 1 | FCZ | 400 | 4000 | 16000 |
| 2 | VCZ | 100 | 1000 | 4000 |
| 3 | CPF | 200 | 2000 | 8000 |
| 4 | OVCZ | 100 | 1000 | 4000 |
| 5 | PCZ | 50 | 500 | 2000 |
| 6 | ICZ | 100 | 1000 | 4000 |
| 7 | HICZ | 100 | 1000 | 4000 |
(4) Sample processing
1) Pretreatment of a standard product: taking 50 mu L of each calibrator sample, putting the 50 mu L of each calibrator sample into a 1.5mL centrifuge tube, adding 200 mu L of protein precipitant containing an internal standard (the volume ratio of methanol to acetonitrile is 1:4), and oscillating at a high speed for 5 min; centrifuging at 14000r/min at 4 ℃ for 5 min; transfer 60. mu.L of supernatant from EP tubing to plastic lined tubing in a 1. mu.L sample size
2) Pretreatment of a serum sample: putting 50 μ L of serum into a 1.5mL centrifuge tube, adding 200 μ L of protein precipitant containing internal standard (volume ratio of methanol to acetonitrile is 1:4), and shaking at high speed for 5 min; centrifuging at 14000r/min at 4 ℃ for 5 min; transfer 60. mu.L of supernatant from the EP tube to a plastic lined tube in a 1. mu.L sample volume.
3) Pretreatment of quality control products: the quality control solutions QC (L), QC (M), QC (H) are respectively taken and 50 μ L of each quality control solution QC (L), QC (M), QC (H) are respectively put into a 1.5mL centrifuge tube, and then the quality control solutions QC (L), QC (M), QC (H) are consistent with the pretreatment of the serum sample, and the details are not.
The components of the assay kit are shown in Table 8.
TABLE 8 preparation of kit Components for analysis of antituberculosis drugs (100 persons)
Remarking: the protein precipitant containing the internal standard is prepared by the following method, 200 mu L of the mixed internal standard solution is added into 19.8mL of protein precipitant to obtain the protein precipitant containing the internal standard. The protein precipitant containing the internal standard is preferably prepared as it is.
Fourth, method verification
1. Extracting an ion current chromatogram: the antifungal drug standard and the serum sample have symmetrical peak shapes and no interference of miscellaneous peaks, which indicates that the antifungal drug standard can be well detected under the conditions, and fig. 1 is an extracted ion current chromatogram of the antifungal drug standard; FIG. 2 is an extracted ion flow chromatogram of an antifungal agent in a serum sample.
2. Calibration curve: and establishing a calibration curve by adopting an isotope internal standard quantitative method and utilizing TargetLynx software to calculate the concentration of the substance to be detected in the serum by taking the concentration ratio of the standard substance to the internal standard substance as an X axis and the peak area ratio of the standard substance to the internal standard substance as a Y axis. The linear fitting equation of the antifungal drugs in the respective concentration ranges has good linearity, the correlation coefficient is more than 0.99, and the quantitative requirements are met, which is shown in Table 9.
TABLE 9 Linear regression equation and Linear correlation coefficient for antifungal drugs
| Serial number | Compound (I) | Retention time (min) | Linear range (ng/mL) | Linear equation of equations | Coefficient of correlation (r) |
| 1 | FCZ | 2.16 | 200-40000 | y＝0.000167598*x-0.00990376 | 0.9984 |
| 2 | VCZ | 2.87 | 50-10000 | y＝0.00156148*x-0.00375731 | 0.9994 |
| 3 | OVCZ | 2.46 | 50-10000 | y＝0.00065887*x-0.00224909 | 0.9985 |
| 4 | PCZ | 3.11 | 25-5000 | y＝0.0115402*x+0.0336516 | 0.9997 |
| 5 | ICZ | 3.56 | 50-10000 | y＝0.00340923*x-0.0565839 | 0.9965 |
| 6 | HICZ | 3.17 | 50-10000 | y＝0.000804196*x-0.00657326 | 0.9999 |
| 7 | CPF | 2.37 | 100-20000 | y＝0.000554567*x-0.00443162 | 0.9986 |
3. Accuracy survey: and evaluating the accuracy of the method by adopting a standard recovery rate test. A mixed blank serum sample is prepared, low, medium and high 3 concentrations of standard substances are respectively added, the treatment is repeated by the same steps and the measurement is carried out for 5 times, the result shows that the adding standard recovery rate of the antifungal medicine is between 86.35 and 112.10 percent, the RSD of 5 times of repeated tests is in the range of 1.34 to 7.81 percent, and the statistical result is shown in the table 10.
TABLE 10 results of recovery of antifungal drug spiked
4. And (3) precision test: taking an interference-free blank serum sample, adding antifungal drug standards with different concentrations to obtain serum samples with low, medium and high concentrations, repeatedly treating 6 batches in one day for three days continuously, quantitatively determining the concentration of the antifungal drug by an isotope internal standard method, wherein the in-batch precision is 2.44-8.14%, treating 3 batches in three days, and calculating the inter-batch precision to be 2.95-7.54%, and the result is shown in Table 11.
Fifth, discuss
The invention establishes a method for simultaneously measuring antifungal drugs in human serum by using ID-UPLC-MS/MS. The serum dosage is less (only 50 mu L), the pretreatment is simple, and the analysis of various substances by one injection only needs 5min, and the method is simple and quick.
The isotope internal standard method is adopted for quantification, so that the matrix interference can be greatly eliminated, the result is not influenced by conditions such as a pretreatment process, instrument response fluctuation and the like, and accurate quantification can be achieved. The accuracy of the method is evaluated by a standard adding recovery rate test, and the result shows that the standard adding recovery rate of the antifungal medicine is between 86.35 and 112.10 percent, the RSD of 5 times of repeated tests is 1.34 to 7.81 percent, and the accuracy is good.
The reproducibility result of the method shows that the precision of the antifungal medicament in batches is 2.44-8.14%, the precision between batches is 2.95-7.54%, and the method has good reproducibility.
In a word, the method has the advantages of high sensitivity, strong specificity, accuracy and simple pretreatment process, completes the separation and detection of the compound within 5min, meets the requirements on accuracy and precision, can be used for quantitative analysis of clinical serum antifungal drugs, and provides a reliable detection method for monitoring related drug concentrations.
The above examples are only intended to illustrate the technical solution of the present invention, but not to limit it; although the present invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: modifications of the technical solutions described in the foregoing embodiments are still possible, or some technical features may be equivalently replaced; and the modifications or the substitutions do not make the essence of the corresponding technical solutions depart from the scope of the technical solutions of the embodiments of the present invention.
Claims (8)
1. A reagent kit for detecting antifungal drugs in serum by an ultra-high performance liquid chromatography tandem mass spectrometry technology,
the antifungal drugs are respectively: FCZ, VCZ, OVCZ, PCZ, ICZ, HICZ, CPF.
The kit is characterized by comprising the following reagents:
(1) eluent:
mobile phase A: 0.01 to 0.2 percent of formic acid aqueous solution, and a mobile phase B: acetonitrile;
(2) a calibration sample:
calibrator samples at seven different concentration points S7-S1:
s7: contains CPF 20000ng/mL, VCZ/OVCZ/ICZ/HICZ 10000ng/mL, FCZ 40000ng/mL, PCZ5000 ng/mL;
s6: contains 10000ng/mL CPF, 5000 VCZ/OVCZ/ICZ/HICZ, 20000ng/mL FCZ and 2500ng/mL PCZ;
s5: contains CPF 5000ng/mL, VCZ/OVCZ/ICZ/HICZ 2500, FCZ 10000ng/mL, PCZ 1250 ng/mL;
s4: contains CPF1000 ng/mL, VCZ/OVCZ/ICZ/HICZ 500, FCZ 2000ng/mL, PCZ 250 ng/mL;
s3: contains CPF 500ng/mL, VCZ/OVCZ/ICZ/HICZ 250, FCZ 1000ng/mL, PCZ 125 ng/mL;
s2: contains 200ng/mL CPF, 200ng/mL VCZ/OVCZ/ICZ/HICZ 100, 400ng/mL FCZ and 50ng/mL PCZ;
s1: contains CPF100 ng/mL, VCZ/OVCZ/ICZ/HICZ 50, FCZ 200ng/mL, PCZ 25 ng/mL;
(3) mixing internal standard solutions:
comprises methanol water solution of FCZ-d44000ng/mL, VCZ-d31000ng/mL, CPF-d42000ng/mL, OVCZ-d31000ng/mL, PCZ-d 42000ng/mL, ICZ-d 55000 ng/mL and HICZ-d 54000 ng/mL;
(4) protein precipitant:
a mixed solution of methanol and acetonitrile;
(5) quality control product:
the serum matrix solution containing antifungal drugs is divided into low, medium and high concentrations, namely QC (L), QC (M) and QC (H), wherein the QC (L) comprises the following components: FCZ 400ng/mL, VCZ 100ng/mL, CPF 200ng/mL, OVCZ 100ng/mL, PCZ50ng/mL, ICZ 100ng/mL, HICZ 100 ng/mL;
QC (M) comprises: FCZ 4000ng/mL, VCZ 1000ng/mL, CPF 2000ng/mL, OVCZ 1000ng/mL, PCZ500ng/mL, ICZ 1000ng/mL, HICZ 1000 ng/mL;
QC (H) includes: FCZ 16000ng/mL, VCZ 4000ng/mL, CPF 8000ng/mL, OVCZ 4000ng/mL, PCZ2000ng/mL, ICZ 4000ng/mL, HICZ 4000 ng/mL.
2. A reagent kit for detecting antifungal drugs in serum by an ultra-high performance liquid chromatography tandem mass spectrometry technology,
the antifungal drugs are respectively: FCZ, VCZ, OVCZ, PCZ, ICZ, HICZ, CPF.
The kit is characterized by comprising the following reagents:
(1) eluent:
mobile phase A: 0.01 to 0.2 percent of formic acid aqueous solution, and a mobile phase B: acetonitrile;
(2) a calibration sample:
mixed standard solution containing FCZ 800000ng/mL, VCZ 200000ng/mL, CPF 400000ng/mL, OVCZ 200000ng/mL, PCZ100000ng/mL, ICZ200000ng/mL, HICZ200000ng/mL and blank serum matrix; before detection, calibration sample samples S7-S1 at seven different concentration points and quality control solution QC (L), QC (M) and QC (H) at three concentrations of low, medium and high are prepared by dilution according to the following formula recorded in an instruction book:
s7: contains CPF 20000ng/mL, VCZ/OVCZ/ICZ/HICZ 10000ng/mL, FCZ 40000ng/mL, PCZ5000 ng/mL;
s6: contains 10000ng/mL CPF, 5000 VCZ/OVCZ/ICZ/HICZ, 20000ng/mL FCZ and 2500ng/mL PCZ;
s5: contains CPF 5000ng/mL, VCZ/OVCZ/ICZ/HICZ 2500, FCZ 10000ng/mL, PCZ 1250 ng/mL;
s4: contains CPF1000 ng/mL, VCZ/OVCZ/ICZ/HICZ 500, FCZ 2000ng/mL, PCZ 250 ng/mL;
s3: contains CPF 500ng/mL, VCZ/OVCZ/ICZ/HICZ 250, FCZ 1000ng/mL, PCZ 125 ng/mL;
s2: contains 200ng/mL CPF, 200ng/mL VCZ/OVCZ/ICZ/HICZ 100, 400ng/mL FCZ and 50ng/mL PCZ;
s1: contains CPF100 ng/mL, VCZ/OVCZ/ICZ/HICZ 50, FCZ 200ng/mL, PCZ 25 ng/mL;
QC (L) includes: FCZ 400ng/mL, VCZ 100ng/mL, CPF 200ng/mL, OVCZ 100ng/mL, PCZ50ng/mL, ICZ 100ng/mL, HICZ 100 ng/mL;
QC (M) comprises: FCZ 4000ng/mL, VCZ 1000ng/mL, CPF 2000ng/mL, OVCZ 1000ng/mL, PCZ500ng/mL, ICZ 1000ng/mL, HICZ 1000 ng/mL;
QC (H) includes: FCZ 16000ng/mL, VCZ 4000ng/mL, CPF 8000ng/mL, OVCZ 4000ng/mL, PCZ2000ng/mL, ICZ 4000ng/mL, HICZ 4000 ng/mL;
(3) mixing internal standard solutions:
comprises methanol water solution of FCZ-d44000ng/mL, VCZ-d31000ng/mL, CPF-d42000ng/mL, OVCZ-d31000ng/mL, PCZ-d 42000ng/mL, ICZ-d 55000 ng/mL and HICZ-d 54000 ng/mL;
(4) protein precipitant:
a mixed solution of methanol and acetonitrile;
(5) the use instruction comprises: comprises calibrator samples S1-S7 and quality control solution QC (L), QC (M) and QC (H) with low, medium and high concentrations.
3. The kit according to claim 1 or 2,
the mobile phase A is 0.01-0.2% aqueous formic acid solution, preferably 0.1% aqueous formic acid solution.
4. The kit according to claim 1 or 2,
the volume ratio of methanol to acetonitrile in the protein precipitator is 1: 1-5; preferably, the volume ratio of methanol to acetonitrile in the protein precipitant is 1: 4.
5. The kit of claim 1 or 2, wherein the non-interfering serum substrate is a blank serum free of antifungal agents.
6. The kit according to claim 1 or 2,
the kit also comprises a chromatographic column Agilent Zorbax Eclipse XDB C18 with the specification of 3X 100mm and 1.8 μm.
7. The kit according to claim 2,
the preparation method of the quality control solution QC (L), QC (M) and QC (H) with low, medium and high concentrations described in the specification is as follows:
QC (L) is QC (M) and is diluted to 10 times by using an interference-free serum substrate;
QC (M) is the mixed standard solution diluted to 200 times by using an interference-free serum substrate;
QC (H) is the above mixed standard solution diluted 50-fold with non-interfering serum base.
8. The use of the kit of any one of claims 1-2 for detecting antifungal drugs in serum by ultra high performance liquid chromatography tandem mass spectrometry.
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| CN115219611A (en) * | 2022-03-10 | 2022-10-21 | 上海义准生物有限公司 | Kit for determining 7 antifungal drugs in serum by ultra-high performance liquid chromatography-tandem mass spectrometry and application thereof |
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| CN115219611A (en) * | 2022-03-10 | 2022-10-21 | 上海义准生物有限公司 | Kit for determining 7 antifungal drugs in serum by ultra-high performance liquid chromatography-tandem mass spectrometry and application thereof |
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