Development, validation, and clinical application of an FIA‐MS/MS method for the quantification of lysophosphatidylcholines in dried blood spots

Abstract Background Lysophosphatidylcholine (LPC) plays pivotal roles in several physiological processes and their disturbances are closely associated with various disorders. In this study, we described the development and validation of a reliable and simple flow injection analysis–tandem mass spectrometry (FIA‐MS/MS)‐based method using dried blood spots (DBS) for quantification of four individual LPC (C20:0, C22:0, C24:0, and C26:0). Methods Lysophosphatidylcholines were extracted from 3.2 mm DBS with 85% methanol containing 60 ng/ml internal standard using a rapid (30 min) and simple procedure. The analytes and the internal standard were directly measured by triple quadrupole tandem mass spectrometry in multiple reactions monitoring mode via positive electrospray ionization. Results Method validation results showed good linearity ranging from 50 to 2000 ng/ml for each LPC. Intra‐ and inter‐day precision and accuracy were within the acceptable limits at four quality control levels. Recovery was from 70.5% to 107.0%, and all analytes in DBS were stable under assay conditions (24 h at room temperature and 72 h in autosampler). The validated method was successfully applied to assessment of C20:0‐C26:0LPCs in 1900 Chinese neonates. C26:0‐LPC levels in this study were consistent with previously published values. Conclusion We propose a simple FIA‐MS/MS method for analyzing C20:0‐C26:0LPCs in DBS, which can be used for first‐tier screening.

generally required to be drawn by venipuncture, while it poses an obvious challenging task to implement in the pediatric populations.
In addition to collecting venous blood, dried blood spot (DBS) technique offers a more convenient and less-invasive option where a small volume of capillary blood (<100 µl) is acquired to a filter paper by a finger prick without massive blood collection. 9,10 Some of the advantages, such as minimal invasiveness, simplified sample collection, as well as reduced blood volume requirement, facilitate DBS to be most popular among clinics, especially in the laboratory workup for pediatric population. 11,12 Despite the potential benefits of DBS sampling, highly sensitive analytical methods are required for analysis to overcome the limitation from the small size of the samples.
The common technique adopted in DBS is flow injection analysistandem mass spectrometry (FIA-MS/MS). FIA-MS/MS is a simple and fast method, in which samples are introduced to the MS instrument directly without including any chromatographic system. 13,14 This allows the rapid measurement of multiple analytes with high sensitivity and specificity. In view of this, FIA-MS/MS combined with DBS sampling has been rated as one of the most cost-and time-effective strategies and widely utilized for high-throughput Tian's work adopting the NeoBase TM 2 Non-derivatized MSMS kit, no further reported was published on DBS-based assay for measuring LPCs with Chinese neonates. 16 NeoBase TM 2 kit was an upgraded version of NeoBase TM 1 and it added more metabolites including 3 amino acids, 5 acylcarnitines, 4 lysophospholipids, 2 nucleosides, and the coverage was expanded to 57 metabolites.
Although the use of commercial kit might be preferable for simultaneous quantification of various metabolites in clinical laboratories, it would be costly when measuring a portion of targeted compounds.
The aim of this study was to develop a first-tier method for simultaneous and high-throughput quantification of four LPCs (C20:0, C22:0, C24:0, and C26:0) by combining DBS sampling with FIA-MS/ MS. After the bioanalytical validation, the method was applied to a collection of clinical samples of 1900 Chinese newborns to further explore its clinical utility.

| DBS preparation
Independent primary stock solutions of four LPCs were prepared at a concentration of 1 mg/ml in methanol. Primary stock solutions in methanol were then mixed to prepare pooled standard stock solution and stored at −20℃. Working solutions for calibration standards were obtained by diluting the standard stock solution, resulting in a series of solutions with concentrations of 2500, 6250, 12,500, 25,000, 50,000, and 100,000 ng/ml for LPCs. DBS for calibration standards and QC samples were prepared as described below.
Calibration standards were obtained by diluting the working standard 50-fold with pooled EDTA blood to the concentrations between 50 to 2000 ng/ml. The QC samples at 50, 125, 500, and 1000 ng/ ml were also prepared similarly. An aliquot (50 µl) of spiked blood was subsequently spotted on filter paper card and dried overnight at room temperature. All the DBS cards were finally stored at −20℃ in ziplock bags with desiccant.

| Method validation
Linearity of the assays was assessed from a calibration curve established by plotting the peak-area ratios of analyte to IS vs. the concentration ranging from 50 to 2000 ng/ml. The calibration function was fitted by linear regression model and the coefficient of determination (R 2 ) was calculated.
Accuracy and precision were evaluated by repeatedly analyzing QC samples at four concentration levels with 10 replicates on the same day (intra-day) and between three consecutive days (inter-day).
Accuracy was calculated from the percent difference between the  To investigate the effects of the hematocrit (Hct) of whole blood on quantification, fresh blood was centrifuged and the red blood cells were washed three times with saline. Then, different volumes of plasma and red blood cells were mixed to obtain blood samples with different Hct levels (0.30, 0.40, 0.5, and 0.6). QC concentrations at 50, 125, 500, and 1000 ng/ml were tested at four Hct Levels.

| Application to clinical samples
This study was reviewed and approved by Beijing Obstetrics and Gynecology Hospital Research Ethics Committee. A total of 1900 anonymous samples were obtained from residual newborn DBS samples following routine newborn screening between April 2020 and June 2020 in Beijing Newborn Screening Center. All participants were negative for screening programs. DBS were collected by heel prick method between 24 h and 7 days after birth and spotted on Whatman filter paper cards for analysis. In addition, a shaking incubation step was included because of its reduced extraction time and improved extraction efficiency.

| Statistical analysis
Experiments were also conducted to determine optimal incubation conditions by testing different incubation time (30, 45, and 60 min), incubation temperature (room temperature, 45℃), and shaking speed (450, 750 rpm). No significant difference was observed with respect to the extraction efficiency. Nevertheless, 45℃ was selected as the optimal temperature because it is easy to control. Therefore, the optimal values regarding incubation procedure were 30 min, 45℃, and 450 rpm.

| FIA-MS/MS conditions
Subsequently, the elution conditions were further investigated. For the mobile phase composition, methanol, the mixture of methanol and water was tested, with formic acid (0.05%, 0.1%), ammonium acetate (5 mM) used as an additive under all conditions. It was found that addition of ammonium acetate could provide higher peak intensity than formic acid ( Figure 1). However, when the ammonium acetate and formic acid added together, no extra signal enhancement was observed. Therefore, methanol/water (85/15, v/v) with 5 mM ammonium acetate was selected as the mobile phase.
In terms of the elution gradient, both constant flow rate and variable flow rate elution were evaluated. In the initial stage of method development, analytes in neat solutions were able to acquire symmetrical peaks using constant flow rate; however, chromatographic peak shapes from DBS extraction became bifurcation and tailing. It may be due to the reason that the entire sample injection band is introduced at the same time to the mass spectrometer in the FIA-MS/ MS run; thus, analytes of interest are not separated from endogenous compounds which could lead to interferences. 13,18 Consequently, we switched to variable flow rate that started with a rapid flow rate followed by a reduced rate to obtain a wide chromatographic peak and form a relative steady state for analysis.  The methods were evaluated in terms of intra-day accuracy and precision by assaying QC samples. As shown in Table 1, the intraday precision for all compounds was less than 14% and accuracy ranges from 85.0% to 115.8% (Table 1). The inter-day precision was assessed by analyzing three sets of all QC samples on three consecutive days. The inter-day CV was less than 16% with the corresponding accuracy of 85.4%-115% (Table 1). This method displayed satisfactory precision and accuracy meeting acceptance criteria for bioanalytical methods.

| Method performance
Recovery was determined by analyzing DBS samples prepared from pooled blood with known analyte concentrations before and after the spiking of the four levels of QC standards. In this study, the recovery was 88.4%-106.83% at the concentrations of 125, 500, and 1000 ng/ml ( Table 2), but lower than 80% at the concentration of 50 ng/ml owing to the endogenous levels are higher than the spiked concentration.
The stability of LPCs before and after pretreatment samples was tested under a variety of conditions using QC samples. The stability results (Table S1) showed that no significant degradation of analytes in DBS samples was observed when stored at room temperature for 24 h. Additionally, the stability of processed sample stability in the  (Table S2). In summary, limited effect of hematocrit on LPCs concentration in DBS samples was observed.

| Clinical application
Next, the developed method was applied to 1900 DBS samples from

| CON CLUS IONS
In this study, we described a fast, reliable, and simple method for determination of LPCs in DBS by FIA-MS/MS with ESI positive ionization. The method allowed the analysis of maximum thirty samples per hour. The straightforward samples preparation procedure can be applied in a high throughput manner. In addition, DBS sampling provided more convenient and comfortable way for special populations such as critically ill and infants. In this way, it was beneficial to analyze the vast majority of samples in the clinical practice with this method, which was time-and cost-effective. The developed method was fully validated in terms of the linear range, accuracy, precision, recovery, stability, and Hct and has been successfully applied to the real sample analysis. In view of the pivotal roles of LPCs in various diseases, we believe that the utility of the method is not limited to X-ALD newborn screening application but also has potential to be extended to predict the severity of many others. However, more rigorous clinical evaluation of the present method needs to be carried out in the future to further verify its utility in various clinical scenarios.

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.