Applicability of Vancomycin, Meropenem and Linezolid in capillary microsamples versus dried blood spots: a validation and pilot study suitable for microsampling in critically ill children

doi:10.21203/rs.3.rs-2010514/v1

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Applicability of Vancomycin, Meropenem and Linezolid in capillary microsamples versus dried blood spots: a validation and pilot study suitable for microsampling in critically ill children

https://doi.org/10.21203/rs.3.rs-2010514/v1

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Therapeutic drug monitoring (TDM) of antimicrobials is extremely useful in critically ill patients. However, it’s a burden for neonate or children with small circulating blood volume. In the present study, we aimed to develop and validate a microsampling TDM platform (including dried blood spots (DBS) and capillary microsamples (CMS)) for the simultaneous quantification of vancomycin, meropenem and linezolid. Paired DBS and CMS samples were obtained from ICU to evaluate its clinical application. Estimated plasma concentrations (EPC) were calculated from DBS concentrations. Agreement between methods was evaluated using Deming regression and Bland–Altman difference plots. Results for microsampling methods validation showed excellent reliability and compatible with the analysis of sample matrix and hematocrit range of the studied population. DBS and CMS results for accuracy and precision were within accepted ranges, samples were stable at room temperature for at least 2 days and 10h, respectively. Hematocrit has no impact on CMS, but sightly impact on DBS measurement. The CMS and DBS antibiotics concentrations correlated well (r > 0.98). The ratio of the drug concentration in DBS samples to that in CMS was 1.39 for vancomycin ,1.34 for meropenem and 0.94 for linezolid. EPC calculated from DBS using individual hematocrit presented comparable absolute values for vancomycin (slope:1.06) and meropenem(slope:1.04), being in mean 98%,99% of the measured CMS concentrations, respectively.

Conclusion: This study provides a microsampling TDM platform validated for clinical use for a rapid quantification of three antibiotics in a small volume of blood and is suitable for real-time TDM-guided personalization of antimicrobial treatment in critically ill children.

Capillary microsampling

DBS

Antibiotics

Pediatric

Agreement

Intensive care units

The small circulating blood volume in critically ill children limited us to monitoring the drug concentration frequently. And microsampling technology might be a solution to achieving the challenging problem.
DBS is a dried-format microsampling techniques have advantages in better analyte-stability but likely be affected by hematocrit. And CMS was called the closest microsample to the ‘gold standard’ of plasma sampling, but it may be influenced by adsorption and dilution.

What is new

Reported the feasibility of microsamping TDM platform for combined quantifying vancomycin, meropenem and linezolid for critically ill children.
Results for microsampling study showed vancomycin, meropenem and linezolid could be accurate determination in CMS. And red blood cells may act a diluent to the plasma for vancomycin and meropenem in DBS measurement.
Critically ill children often have low and variable hematocrit, which cause individual Hct values had better performance than average Hct in measuring DBS concentration.

Therapeutic drug monitoring (TDM) offers great impact to improve treatment efficacy while minimizing toxicity. However, the small circulating blood volume in critically ill children did not allow us to determining the drug concentration frequently [1]. Therefore, microsampling method is urgent need in critically ill children for TDM[2, 3]. The microsampling technologies has included options for wet- or dried-format sampling, such as capillary microsample (CMS) or dried blood spot (DBS), which has implications for storage, transport, determine and accuracy of the microsample[4].

Vancomycin (VAN), meropenem (MEM) and linezolid (LZN) are frequently used in neonate and pediatric intensive care units (ICUs)[5]. However, VAN may cause nephrotoxicity in children[6], LZN demonstrate wide interindividual variability which result in insufficient levels in 63% of patients[7], MEM is a time-dependent drug with rapid metabolism and have risk in treatment failure, toxicity and possible antimicrobial resistance[8]. TDM can be an excellent tool in treatments with those three antibiotics, especially in the case of insufficient evidence for critically ill children.

Previous studies of Barco et al. published an analytical method for simultaneous determining of 14 antibiotics (including VAN, MEM and LZN) in plasma[9] ,But have not dealt with microsampling. Several methods based on DBS or CMS are available in separately determining three antibiotics in the literatures[10–13]. From a clinical standpoint, small-volume sampling and simultaneous determination of three antibiotics could be useful in pediatric intensive care units. And comparative evaluation of the relationship between sampling technology and drug characteristics and may be worth exploring. In this paper, we focus on the quantification of VAN, MEM and LZN in critically ill children used by capillary microsample and dried blood spots.

A first aim of this study was to set up and apply a fully validated microsampling TDM for the simultaneous determination and quantification of three antibiotics in DBS and CMS. Evaluating the reliability of two microsampling methods applied for routine TDM in critically ill children, was the second aim of this study.

2.1 reagents and chemicals

The reference standard of vancomycin (Lot No.130360–202103), meropenem (Lot No.130506) and linezolid (Lot No.130640–201901) were complying with ChP Reference Standards. The internal standard (IS) of norvancomycin (Lot No.130338–201704), meropenem-D6(Lot No.21B063-A5) and linezolid-D3(Lot No.21B035-A1) were purchased from ZZBIO (shanghai, China). Ultrapure water was provided by a smart2pure pro water purification system (Thermo scientific, USA). Acetonitrile (ACN), methanol (MeOH) and formic acid was from Sigma-Aldrich (Shanghai, China). Solvents were all of LC-MS grade. Whatman 903® paper was purchased from GE Healthcare. Capillary tubes (40 µL and 70µL) were purchased from Hirschmann Co. Ltd (Eberstadt, German)

Stock solutions of VAN, MEM and LZN were prepared in MeOH at concentrations of 4,4 and 2 mg/mL, respectively. All stock solutions were stored at − 80°C and were further diluted in respectively 50% MeOH to obtain working solutions. All individual IS stock solutions were prepared in ACN at 1 mg/mL and also stored at − 80°C. The IS working solution consisted of 2.5 µg/mL norvancomycin,5.0 µg/mL meropenem-D6, and 1.0 µg/mL linezolid-D3.

2.2 Calibrators and quality control (QC) samples

Calibrators were prepared at eight concentrations, based on the therapeutic ranges, by spiking working solutions to blank human plasma and whole blood at a concentration of 1, 2, 4, 8, 15, 30, 50 and 100 µg/mL for VAN; 0.4, 0.8, 1.6, 3.2, 6, 12, 20 and 40 µg/mL for MEM; 0.2, 0.4, 0.8, 1.6, 3, 6, 10 and 20 µg/mL for LZN. QC samples (lower limit of quantification (LLOQ), low, medium and high, respectively) were prepared by spiking working solutions to blank plasma and whole blood at 1, 3, 7.5 and 75 µg/mL for VAN; 0.4, 1.2, 3 and 30 µg/mL for MEM; 0.2, 0.6, 1.5 and 15 µg/mL for LZN. The amount of solvent added to matrix, both for calibrators as QCs, did not exceed 5%. Calibrators and QC samples were aliquoted and stored at − 80°C until analysis.

2.3 Sample preparation

All samples were subjected to protein precipitation:

For CMS samples, Briefly, the whole blood samples for patient samples were filled in capillary tubes, and centrifugated for 5 min at 3,000×rpm. The volume of CMS was measured and cut into Eppendorf tube. the cut capillary tube was washed by blank plasma (the plasma volume was replenished to 50 µL), 15 µL formic acid and 50 µL ACN in turn. Subsequently, an aliquot of 50 µL calibrator and QC sample was add in Eppendorf tube, mixed with the same reagents. All samples were added with 150µL IS working solution, shaken at 2000 rpm for 5 min, and centrifugated for 5 min at 12,500×rpm. 10 µL supernatant was transferred and dilute 20-fold in water. Finally, 5µL diluent was injected into the LC-MS/MS for analysis.

For DBS samples, an aliquot of 50 µL whole blood samples for calibration curve, QC and unknown samples were spotted on Whatman 903® paper cards and dried in the shade at ambient temperature for at least 30 min. DBS samples were cut off at 10 mm diameter and transferred into Eppendorf tubes. Then 200 µL of aqueous solution (containing 2% FA, v/v) and 200 µL of IS working solution were added into the tubes. Samples were shaken at 2000 rpm for 20 min at room temperature and for 5 min at 12,500×rpm. The supernatant was dilute and injected as same as CMS.

2.4 Chromatographic analysis

Chromatographic analysis was performed using a ACQUITY UPLC system (waters Corporation, USA). Mobile phases consisting of 0.2% FA in water (A) and ACN (B) at a flow rate of 0.3 mL/min were used. Separation of the components was achieved by gradient elution: the mobile phase B gradient was initiated at 10% B in 0.5 min, then linearly increased to 70% in 2.8 min and to 90% in 3 min, maintained at 90% in 3.3 min, continuously decreased to 10% in 3.5 min, and maintained at 10% until the end of the run, resulting in a total run time of 4 min. The UPLC system was equipped with an Acquity UPLC® HSS T3 column (100 mm length x 2.1 mm i. d., 1.8 µm) (Waters, Milford, MA, USA) fitted with a corresponding BEH C18 guard column, maintained at 40°C. The autosampler temperature was set at 10°C.

The LC system was coupled to Xevo TQ-S Micro triple quadrupole mass spectrometer (Waters Corporation, USA) equipped with an electrospray ionization (ESI) source operating in positive ion mode. Multiple reaction monitoring (MRM) was applied for detection of the components. The desolvation temperature was 550°C, cone gas flow was 150 L/hr, and desolvation gas flow was 900 L/hr. MRM transitions with corresponding MS parameters for quantifiers, qualifiers and ISs are listed in Table 1. All UPLC-MS/MS data were collected and processed by Masslynx 4.1 software (Waters Corp, MA, USA).

Table 1

Retention time, MRM transitions and compound-specific MS settings.
compound	Retention time (min)	Parent ion(m/z)	Daughter ion(m/z)	Cone (V)	Collision
Vancomycin	1.88	725.9	144.2	40	15
Meropenem	2.03	384.02	141.2	25	12
Linezolid	2.75	338.03	296.02	12	18
Norvancomycin	1.82	718	144.2	40	13
Meropenem-D6	2.03	390.2	146.8	12	18
Linezolid-D3	2.75	341.1	297	12	18

Table 2

Results of accuracy and precision for vancomycin, meropenem, and linezolid in plasma, DBS and capillary microsamples.
			Within-run (n = 6)		Between-run (n = 6)			Within-run (n = 6)		Between-run (n = 6)			Within-run (n = 6)		Between-run (n = 6)
Matrix	QC level	Nominal concentration (µg /mL)	CV%	Bias%	CV%	Bias%	Nominal concentration (µg /mL)	CV%	Bias%	CV%	Bias%	Nominal concentration (µg /mL)	CV%	Bias%	CV%	Bias%
Drug		Vancomycin					Meropenem					Linezolid
Plasma	LLOQ	1.00	3.7	-9.8	4.0	-6.4	0.400	5.6	-4.0	4.5	-2.2	0.200	3.7	-0.4	3.3	2.3
	Low	3.00	4.0	12.7	4.0	12.3	1.20	2.8	-2.6	4.3	-4.8	0.600	1.2	5.4	2.7	6.5
	Medium	7.50	1.8	6.7	3.7	9.0	3.00	1.3	0.4	3.9	-2.5	1.50	1.1	3.4	1.5	2.9
	High	75.0	1.4	-5.7	4.0	-1.7	30.0	1.3	-5.8	3.1	-6.7	15.0	1.9	-5.9	2.4	-6.8
DBS	LLOQ	1.00	4.5	6.1	3.7	6.2	0.400	4.7	9.1	9.8	8.0	0.200	2.3	4.4	2.7	4.0
	Low	3.00	2.3	1.6	3.2	1.9	1.20	2.3	7.3	5.2	4.7	0.600	1.1	5.0	1.4	4.8
	Medium	7.50	1.5	-2.8	3.4	-5.6	3.00	1.1	-2.6	7.3	-9.1	1.50	0.7	-6.6	2.6	-8.8
	High	75.0	2.8	-0.5	3.3	-0.1	30.0	2.1	5.5	3.8	2.1	15.0	2.9	1.1	2.7	-0.7

2.5 Method validation

The method validation was followed the guidance of Chinese Pharmacopeia guidance and bioanalytical method validation guidance from ICH. Specifically, selectivity was evaluated by endogenous interference peaks at the retention time of VAN, MEM, and LZN and IS of blank samples from six individuals of whole blood samples. Linearity was validated by calibration curves. Peak area ratios to IS were taken as the ordinate and the nominated concentrations of three antibiotics were taken as the abscissa, which were fitted to 1/x² weighted linear regression. For the intra and inter batch precision and accuracy, six replicates of QC samples and LLOQ samples were determined at three analytical batches. Accuracy was accepted within the range of 85–115% while the coefficient of variance (CV) of precision was below 15%. The matrix effects were carried out at low, medium and high concentrations. Blank plasma and DBS samples from six different individuals were prepared and then added working solution to the extractant in order to evaluate the matrix effects. Stability of plasma and DBS samples at different storage conditions were evaluated. Samples stored at room temperature, 2 ~ 8°C, 35°C, -80°C, and post-treated stability at autosampler were evaluated by examining two levels of QC samples (low and high). Samples were considered stable if concentrations were within the range of 85–115%.

For the accuracy and adsorption test of CMS, six replicates of QC samples were determined. The impact of the Hct on antibiotics DBS measurements was also tested. Blood samples with %Hct of 15,20,25, 30, 35 and 40 were obtained by whole blood and then adding or removing plasma. QCL and QCH DBS samples were prepared from the blood and analyzed in triplicate at fixed Hct level of 30%. The influence of the Hct% on VAN, MEM and LZN determinations was expressed as the percentages of nominal concentrations measured in DBS. Acceptance criteria were values between 85 and 115%.

2.6 DBS and CMS method comparison

From the drug concentration in DBS and the hematocrit (Hct), an estimated plasma concentration (EPC) was calculated from the using the formula \(\text{E}\text{P}\text{C}=\frac{{C}_{DBS}}{1 - (\text{H}\text{c}\text{t}/100)}\). EPC was evaluated both using individual and the average Hct of the group of patients. Agreement between methods was evaluated using Deming regression and Bland–Altman difference plot. Statistical analyses were performed with R4.04 software.

2.7 Clinical applications

A large whole blood sample set were drawn for routine TDM purpose from pediatric patients receiving VAN, MEM and LZN for the treatment of various infections at the intensive care unit of the children’s Hospital of Fudan University, Shanghai, China. The patients were not selected in advance. Samples were included as they were received in our laboratory for routine TDM. DBS and CMS were produced from these whole blood samples. The samples were maintained at -80 ℃ until analysis.

3.1 Method validation of capillary plasma and DBS method

Typical chromatograms obtained from plasma and DBS of VAN, MEM and LZN in blank, blank with internal standard and LLOQ are illustrated in Figure. 1.

There was no endogenous substances interference between analytes and internal standard. Linearity was acceptable using a 1/x² weighting over a range of 1 ~ 100 µg/mL for VAN, 0.4 ~ 40 µg/mL for MEM and 0.2 ~ 20µg/mL for LZN. The two matrixes (plasma and DBS) results of the intra- and inter- assay precision and accuracy were all inside the acceptable ranges (Table.2). The matrix effects for three antibiotics at low, medium and high concentrations were full complied with the testing criteria (Table 3).

Table 3

Results of matrix effect for vancomycin, meropenem, and linezolid in plasma and DBS (n = 6).
Matrix	QC level	Vancomycin		Meropenem		Linezolid
		mean%	SD%	mean%	SD%	mean%	SD%
plasma	Low	98.3	2.1	109.0	3.1	118.5	2.9
	Medium	98.9	0.7	108.3	3.2	117.3	1.8
	High	100.3	1.5	111.9	2.7	109.4	2.0
DBS	Low	98.7	3.0	91.1	3.0	95.6	1.5
	Medium	94.8	3.5	94.1	6.0	90.8	2.1
	High	99.0	2.6	94.2	5.7	94.8	2.8

Table 4

Results of stability for vancomycin, meropenem, and linezolid in plasma and DBS (n = 3).
DBS
Drug	Vancomycin			Meropenem			Linezolid
Condition	4℃	RT	35℃	4℃	RT	35℃	4℃	RT	35℃
Period	3 days	3 days	3 days	2 days	2 days	1 day	3 days	3 days	3 days
QCL	6.6	-5.0	-12.2	2.9	-0.7	-7.4	-7.4	-6.4	-10.1
QCH	-2.3	0.5	2.1	-2.5	-8.9	-9.9	-1.7	-0.5	-6.3
Plasma
Drug	Vancomycin			Meropenem			Linezolid
Condition	4℃	RT	-80℃	4℃	RT	-80℃	4℃	RT	-80℃
Period	1 day	1 day	214 days	1 day	10 h	70days	1 day	1 day	199 days
QCL	6.0	-10.2	-11.1	-8.2	0.4	-7.5	-4.3	-11.8	-2.6
QCH	--5.7	1.5	10.6	-4.6	-14.8	-6.9	-5.5	0.0	2.8

The results of the stability testing of VAN, MEM and LZN in plasma were shown in Table. 2. VAN and LZN in plasma were stable at 2 ~ 8℃ for 24 h, at -80 ℃ for at least 179 days and at room temperature for 1 days. However, MEM in plasma were stable at 2 ~ 8 ℃ for 24 h, at -80 ℃ for 70 days and at room temperature for 10h. Three consecutive freeze-thaw cycles didn’t show any significant degradation for all three antibiotics. DBS cards were dried and stored in sealed bags showed that VAN and LZN can be considered stable for at least 3 days in DBS at both concentration levels (at 4 ~ 8 ℃, 35℃ and room temperature), and at -80℃ for 73 days. In contrast, MEM showed a significant decay at 35 ℃ storage temperature in DBS for 2 days and less than 15% loss over 30 h storage time. During storage at 4 ~ 8 ℃ and room temperature, MEM was stable in stabilized plasma for 2 days. Similarly, MEM stored at -80 ℃were stable for 73 days in DBS.

The adsorption test on capillary tube and the Hct effect on DBS to perform the accuracy of determinations of VAN, MEM and LZN were shown in Figure. 2. The accuracy of CMS showed a non-significant trend in the range of 95.7 ~ 110.1%, which demonstrated three antibiotics have unspecific surface adsorption to the capillary tube. Therefore, capillary tube could be deemed as plasma separator tube in this study. Conversely, the trend of accuracy bias in DBS caused by sample Hct was obvious, the accuracy bias in DBS was reported cause by blood viscosity. Although accuracy was systematic biased, the results still remained inside of acceptance range in Hct 15 ~ 40% with bias from − 5.0 ~ 14.9% for VAN, -10.7 ~ 13.6% for MEM and − 3.8 ~ 12.7% for LZN. Therefore, results obtained with calibration curves prepared with whole blood at Hct 30% are accurate to quantify antibiotics in DBS from patients with Hct between 15 and 40% with acceptable systematic bias.

3.2 Clinical application and method comparison

After validating the pretreatment and analysis workflow of samples, the microsampling methods was successfully applied on a total of 130 Paired CMS and DBS samples from 97 pediatric patients, obtained from neonatal or pediatric ICU, and Measured and calculated concentrations are presented in tables 5. Three antibiotics concentrations in DBS and CMS were significantly correlated (r > 0.98, p < 0.01). However, CMS to DBS drug concentration ratios were highly variable. VAN was ranging from 1.03 to 1.95 and with an average of 1.39, MEM was 1.34 (1.04 ~ 1.93) and LZN was 0.94(0.77 ~ 1.08), which indicating that LZN could be accurate determination in DBS card (Deming regression: y = 0.99x-0.32), but VAN and MEM concentration in DBS shown a significate bias from that of CMS. We suspected that red blood cells may act a diluent to the plasma for VAN and MEM. Therefore, EPC from DBS measurements were necessary to evaluated.

Tables 5. characteristics of Paired microsamples

Characteristics	Vancomycin	Meropenem	Linezolid
age, years, median (min/max)	4.9(0.05/15)	4.2(0.07/13)	1.8(0.17/8)
source of samples, N,(PICU/NICU)	46/16	47/6	11/5
hematocrit,%, mean(min/max)	29.5(17.7/38.8)	25.8(17.3/34.5)	26.1(20.4/39.0)
plasma concentration, µg/mL, mean(min/max)	11.86(1.35/77.73)	10.77(0.68/41.11)	5.59(0.67/14.91)
DBS concentration, µg/mL, mean(min/max)	8.51(1.02/52.69)	8.15(0.41/34.99)	6.37(0.71/13.79)
plasma/DBS ratio, mean(min/max)	1.39(1.03/1.95)	1.34(1.04/1.93)	0.94(0.77/1.08)
plasma/individual EPC ratio, mean(min/max)	0.98(0.77/1.34)	0.99(0.78/1.34)	/
plasma/average EPC ratio, mean(min/max)	0.98(0.72/1.37)	0.99(0.77/1.43)	/

Critically ill children can commonly experience anemia and hemodilution from medical interventions. The average Hct in this study was significantly lower than normal children (17 ~ 39% VS 35 ~ 45%). Deming regression and Bland-Altman difference of concentrations of VAN and MEM obtained in CMS and EPC were compared, and concentrations of LZN also displayed in Figure. 3. Whether EPC was estimated using individual or average Hct, VAN and MEM exhibits a high degree of agreement ((r > 0.98, p < 0.01), the slope value from Deming regression were close to 1.00 (1.04 VS 1.06, p < 0.01) for VAN, and (1.04 VS 1.04, p < 0.01) for MEM. However, EPC estimated using individual Hct does a better job of fitting than using average Hct by Bland–Altman plots. As shown in the diagram, 83.8% (52/62)of the VAN EPC estimated using individual Hct did not differ by more than 20% from CMS, when compared to which 77.4% (48/62) ) using average Hct. As well as MEM EPC showing the same trend (92.5% (49/53) VS 84.9% (45/53)).

In this work we developed a microsampling TDM platform for the simultaneous quantification of empirical antibiotics commonly prescribed in pediatric critically ill children. 72.9% (43/59) pediatric patients in our study received MEM treatment in combination with VAN or LZN. Microsampling and combined detection appear to be to reduce blood volume from patients, and provide concentrations of multiple drugs. Differed from outpatients, critically ill children were often inserted in a peripherally inserted central catheter, therefore venous microsamples could be feasible in ICU.

We showed that the VAN and MEM concentrations difference in DBS and CMS can very well be explained by the formula based on Hct. However, LZN showed a good agreement between DBS and CMS, this result is similar to that found in previous study[14], this may result in the blood cell-to-plasma partitioning of LZN was 0.855 ± 0.080 (N = 9), the LZN was homogeneously distributed in the blood. Alternatively, CMS was not affected by Hct, and was considered as the closest microsample to the ‘gold standard’ of plasma sampling[4]. Method development about different sample pretreatment on call was conducted to solve the adsorbed problem before validation. We compared the desorption solvent among pure water, acetonitrile, formic acid and plasma, subsequent results showed that blank plasma could extracted antibiotics out from capillary tube, and replenish plasma volume to standard sample preparation.

Critically ill children often have low hematocrit. Red blood cells seem to played a dilution role for some hydrophilic or macromolecule drugs in DBS measurement[15]. However, when this effect was taken into account and an estimated plasma concentration was calculated, individual Hct had better performance than average Hct values.

In clinical practice, the consideration of costs and sampling generality, DBS and CMS seem superior to specialized sampling technologies[16], such as volumetric absorptive microsampling and solid-phase micro-extraction. DBS and CMS would be acceptable for phlebotomists who can easily get high quality samples. But each technique has its own advantages and disadvantages, DBS perform better in stability and robust to transport, which is suitable for remote community hospital and home-sampling. But the Hct effect may have impact on DBS. Therefore, DBS maybe a good tool for instable or hydrophobic (evenly distributed in the blood) drug, such as MEN, LZN and immunosuppressant[17–19]. CMS is commonly used in children’s hospital, and freedom from Hct effect. However, analyte may be surface-adsorbed in capillary tube. Drug with low adsorption and good stability, such as VAN, is suitable for CMS. Finally, the applicability and evaluation of microsampling methods in TDM platform need farther research in bridging study.

In this study we have developed and validated a microsampling TDM platform for the accurate quantification of VAN, MEM and LZN. CMS and DBS sampling can be used for TDM and personalization of antibiotic therapy in severe critically ill children or even neonate.

Funding: This work was supported by the Key Development Program of Children's Hospital of Fudan University (EK2022ZX01); National Natural Science Foundation of China [81874325]; Science and Technology Commission of Shanghai Municipality [19XD1400900].

Availability of data and material: The datasets used and analyzed during the current study are available from the corresponding author on reasonable request.

Conflict of interest: The authors have no relevant financial or non-financial interests to disclose.

Author contributions：L-ZP and X-XY conceived and designed the study. X-XY and W-JL established the fully validated method and collected the data, W-GF and Z-HM QC the data. X-XY analyze the data, drafted the article and wrote the article. L-ZP and X-H reviewed and edited the article. All authors read and approved the final article.

Ethical approval: This study was in compliance with the Declaration of Helsinki and was approved by the local Ethics Committee of the Children’s Hospital of Fudan University, Shanghai, China [No.(2021)455].

Consent to participate: Written informed consent was waived by the local Ethics Committee of the Children’s Hospital of Fudan University in view of the retrospective nature of the study and all the procedures being performed were part of the routine care.

Consent to Publish: The authors affirm that human research participants provided informed consent for publication.

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No competing interests reported.

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Applicability of Vancomycin, Meropenem and Linezolid in capillary microsamples versus dried blood spots: a validation and pilot study suitable for microsampling in critically ill children

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