Development and validation of an LC‐MS/MS method for quantifying nine antimicrobials in human serum and its application to study the exposure of Chinese pregnant women to antimicrobials

Abstract Background To study the prevalence of the exposure of pregnant women to antimicrobials, a sensitive and reliable liquid chromatography‐tandem mass spectrometry (LC‐MS/MS) method was developed and validated to determine nine antimicrobials, namely sulfadimidine, sulfapyridine, sulfadiazine, sulfathiazole, ofloxacin, ciprofloxacin, norfloxacin, tetracycline, and lincomycin, in human serum. Methods The sample preparation procedure included protein precipitation followed by a cleanup step with solid phase extraction (SPE). Separation was carried out using a CORTECS T3 column (100 × 2.1 mm, 2.7 µm) by gradient elution with a runtime of 8.0 min. Detection was performed on a triple quadruple tandem mass spectrometer with scheduled multiple reaction monitoring (sMRM) in positive ion scan mode. Results The calibration curves were linear over the concentration range of 0.5–50 ng/ml, and the limit of quantitation was between 0.01 and 0.2 ng/ml. For each level of quality control samples, the inter‐ and intra‐assay precision values were less than 12.0%, and the accuracy ranged from 86.1% to 109.0%. No significant matrix effect or carryover was observed. The antimicrobials of interest were stable under all investigated conditions. The validated method was applied to analyze clinical samples from pregnant women in China, and 10 out of 500 samples showed the presence of antimicrobial residues. Moreover, compared with the time‐resolved fluoro‐immunoassay (TRFIA) method, the developed method showed greater sensitivity and specificity. Conclusion This study provides a simple and rapid LC‐MS/MS method for the simultaneous measurement of nine antimicrobials in serum samples, which could be a useful tool in clinical utilization.


| INTRODUC TI ON
As an effective and powerful regimen, antimicrobials have been widely used in humans, animals, and plants for preventing or treating infection, as well as for promoting growth. 1 Consequently, this extensive application has been accompanied by a large amount of antimicrobials disposed into the environment, which may be from pharmaceutical processes or excreted as parent compounds or metabolites due to poor gut absorption or incomplete metabolism. 2,3 These compounds are eventually directly or indirectly introduced into the human body by contaminated soil, food, and water, thus posing a potential risk to public health. 4,5 It is evident that the presence of antimicrobials at low levels is associated with several adverse health issues, such toxic effects, allergic reactions, and the development of bacterial resistance. 6,7 The impacts of antimicrobial exposure on pregnant women would be exacerbated, as antimicrobials could be transmitted from mother to infant via the blood-placenta barrier or through breastfeeding. 8 Antimicrobials can also have an impact on the human intestinal flora. The gut bacterial community has many critical functions, such as aiding in the development of the immune system and the metabolic system 9,10 and in the synthesis of vitamin K.
In the early stage after birth, the development of a stable microbial community is essential. 11 Antimicrobial exposure in infancy would disrupt the microbiome composition in the gut, which would have long-lasting effects on the host physiology. 12 Therefore, it is necessary to develop an analytical method capable of detecting multiclass antimicrobials to better investigate antimicrobial exposure levels in pregnant women.
To date, several analytical methods have been used to detect antimicrobials, including microbiological inhibition assays, immunoassays, biosensors, and liquid chromatography-tandem mass spectrometry (LC-MS/MS), 13,14 among which LC-MS/MS has become a preferred technique due to its high selectivity and sensitivity. However, only a few LC-MS/MS methods available for the comprehensive analysis of a panel of antimicrobials in human samples have been reported. 15,16 Most methods focus on the analysis of one antimicrobial or a single class of antimicrobials. Furthermore, those methods were used for therapeutic drug monitoring (TDM) or for pharmacokinetic studies.
However, the exposure level of antimicrobials is much lower than the drug concentration in blood, and the species count is much higher because medical and veterinary antimicrobials are all included.
To quickly monitor trace antimicrobial exposure levels in pregnant women, we developed a rapid and sensitive multiclass method capable of detecting nine antimicrobials (four sulfonamides, three quinolones, one tetracycline, and one lincosamide) in human serum

| Chemicals and materials
The antimicrobial standards of sulfadimidine, sulfapyridine, sulfadiazine, sulfathiazole, ofloxacin, ciprofloxacin, norfloxacin, tetracycline, and lincomycin were purchased from the National Institutes for Food

| Preparation of calibrators, quality control samples, and internal standards
Stock solutions of the antimicrobials were individually prepared at a concentration of 1 mg/ml either in MeOH or water. An appropriate amount of each stock solution was spiked into 60% (v/v) ACN and diluted with 10% (v/v) ACN to obtain the mixed working solution series. A seven-point calibrator was prepared by spiking mixed working solutions into blank serum, and the final concentration of the calibrator was 0.5, 1, 2.5, 5, 10, 25, and 50 ng/ml for all the analytes. The quality control (QC) samples were prepared at low (1 ng/ml), medium (20 ng/ml), and high (40 ng/ml) levels for each analyte.
The working internal standard solution was prepared as a mixture, and the concentrations used were 1 µg/ml for norfloxacin-d 8 , 0.2 µg/ml for sulfadimidine-d 4 , 0.4 µg/ml for tetracycline-d 6 , and 0.2 µg/ml for lincomycin-d 3 .

| Instrumental analysis
The sample analyses were performed using a Shimadzu LC-20AD HPLC system coupled with an ABSCIEX 5500 triple quadrupole mass spectrometer equipped with an electrospray ionization (ESI) source.
Chromatographic separation was performed with a Waters CORTECS T3 column (100 × 2.1 mm, 2.7 µm). The column temperature was kept at 35°C. The mobile phase was composed of water containing 0.1% formic acid (A) and ACN containing 0.1% formic acid (B). The gradient program was set as follows: 0 min 5% B, 5 min 60% B, 5.5 min 90% B, 5.5 ~ 6.5 min 90% B, and 6.6 ~ 8 min 5% B. The flow rate was set at 0.3 ml/min, and the injection volume was 2 µl.
The MS was operated in positive ion mode with sMRM. The optimized MS parameters for each antimicrobial are summarized in Table 1. The parameters of the source conditions were as follows:

| Method validation
The method validation was performed in accordance with the rec-
The specificity was investigated by evaluating blank human serum samples from six different subjects and checking for interfering peaks on the chromatogram of the analytes and ISs.
A least-squares regression model was used to check the linearity of the method. The linearity was evaluated by building the calibration curves in human serum at seven concentration levels.
Calibration curves were constructed by plotting the theoretical standard concentration vs. the peak area ratio of the standard to the IS. The limit of quantification (LOQ) was determined as the concentration at which the signal-to-noise (S/N) ratio was >10.
Quality control samples prepared at three different concentrations were used to measure the intra-and interday precision and accuracy. The intraday precision was determined by running each QC sample in six replicates on 1 day, and the interday precision was determined by analyzing six replicates of each QC sample on three successive days. The inter-and intraday precision values were expressed as the coefficient of variation (CV). Accuracy was calculated as the measured concentration when compared to the nominal concentration of each QC and expressed as a percentage. The stability studies were performed using spiked serum at low and high QC concentrations (n = 6) under the following conditions: in the autosampler at 4°C for 24 h, or after undergoing three freeze-thaw cycles at −20°C. The stability was evaluated by calculating the difference between the examined samples and the freshly prepared samples.

| Chromatography and mass spectrometry
Regarding the mass spectrometry conditions, the selection of the precursor and product ion was first carried out by direct infusion of standard solutions of each individual target antimicrobial compound. As a common additive reagent, formic acid is often included in the mobile phase to provide protons and yield improved ionization efficiency. 20 The results revealed that 0.1% formic acid will increase the MS response and improve the peak shape, while a formic acid content that is too high has no obvious effect. Consequently, ACN containing 0.1% formic acid was used as the optimal organic phase.
After optimizing the gradient elution program, nine target antimicrobials were finally analyzed in an 8-min chromatographic run with suitable separation and good symmetric peaks. A representative chromatogram showing typical peak shapes for each antimicrobial is presented in Figure 1.  Table 2.
Intraday and interday precision and accuracy were assessed at the three QC levels. Data on accuracy and precision for each analyte in serum are presented in Table 3. The accuracy for all the compounds was between 86.1% and 109.0%. The intra-and interday precisions for all the analytes ranged from 3.70% to 11.5% and 5.18% to 10.3%, within the 15% limit requested. These results indicated that this method gave satisfactory precision and accuracy for all the antimicrobials obtained from human serum.
The matrix effect is caused by coeluting components from patient sera, resulting in ion suppression or enhancement. The matrix effect was evaluated with three levels of QCs. As shown in Table 2, only minor differences were observed between the pure standards and the postextraction spiked samples, indicating little influence of the background signal of serum on analytes. The recovery for each analyte was greater than 60.0% except for ofloxacin and lincomycin ( Table 2). The carryover of all the antibiotics was within the minimum level (< 1%) (data not shown).
The inaccuracy for freeze-thaw samples ranged from −14.8% to 10.8%. The inaccuracy of autosampler stability ranged from −12.8% TA B L E 2 Calibration range, LOQ, matrix effect and recovery of the analytes  (Table S1).
The performance characteristics of the method are summarized above. More than nine antimicrobials were included in the preliminary experiments, but some of them were not successfully validated for several reasons, such as instability or lack of a suitable IS. There is much work that needs to be performed in future work with the aim of analyzing more antimicrobials.

| Application and comparison
The optimized and validated method was applied to screen antimicrobial residues in serum samples of pregnant women recruited from Beijing Obstetrics and Gynecology Hospital. Among the 500 samples, 10 samples were positive for the target antimicrobials, and the overall detection frequency was 2.0% ( Table 4). The detection frequency of the nine antimicrobials varied from 0.2% to 1.0%. Specifically, ofloxacin and sulfadiazine were the most frequently detected antimicrobials in the serum of pregnant women, accounting for 52.1% and 30.4%, respectively. The highest value was above 50 ng/ml. The lowest concentration in serum was found for lincomycin (0.67 ng/ml). At least two antimicrobials were detected simultaneously in some samples, for example, sample 1 sulfapyridine + sulfadiazine +lincomycin.  but this method suffers from poor specificity because of interferences. 22 The LC-MS/MS method utilizes multiple mass-resolving devices to eliminate the chemical background, providing improved quantification capabilities of multiple known compounds as well as the ability to qualitatively analyze unknowns in biological samples. 23 Therefore, the use of tandem mass spectrometry allows for better selectivity and sensitivity than traditional TRFIA. The comparison results further exhibited the advantage of the developed method for use in clinical practice.

| CON CLUS IONS
In this work, a powerful method for the simultaneous analysis of nine antimicrobials in human serum that are most frequently found in environmental or agricultural products was developed and optimized. The proposed method is rapidly performed and is reliable, and it allows the determination of multiclass antimicrobials belonging to the sulfonamide, quinolone, lincosamide, and tetracycline families in a single run within a short time of 8 min. The assay was successfully validated in terms of sensitivity, linearity, accuracy, precision, recovery, carryover, and stability. Serum samples from pregnant women were analyzed to evaluate the applicability of this method. At least one antibiotic was detected in some samples, and the species accounting for the largest proportions were similar to those found in a previous study. Comparisons of the performance in screening antimicrobials with the TRFIA technique strongly support the sensitivity and selectivity of our presented method. These results indicated that the present method is practical and effective and can be used in clinical laboratories to assess antimicrobial residues to protect pregnant women's health.
However, we note the limitations of this work, such as the slightly tedious sample preparation and the insufficient number of antimicrobials. Future studies should include approaches to simplify the sample treatment procedure and incorporate as many antimicrobials as possible.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data during and/or analyzed during the current study are available from the corresponding author on reasonable request.