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. 2020 May 29;15(5):e0233893. doi: 10.1371/journal.pone.0233893

Determination of piperaquine concentration in human plasma and the correlation of capillary versus venous plasma concentrations

Norah Mwebaza 1,#, Vincent Cheah 2,#, Camilla Forsman 2,#, Richard Kajubi 1, Florence Marzan 2, Erika Wallender 2,3, Grant Dorsey 3, Philip J Rosenthal 3, Francesca Aweeka 2, Liusheng Huang 2,*
Editor: Henk D F H Schallig4
PMCID: PMC7259774  PMID: 32470030

Abstract

Background

A considerable challenge in quantification of the antimalarial piperaquine in plasma is carryover of analyte signal between assays. Current intensive pharmacokinetic studies often rely on the merging of venous and capillary sampling. Drug levels in capillary plasma may be different from those in venous plasma, Thus, correlation between capillary and venous drug levels needs to be established.

Methods

Liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) was used to develop the method. Piperaquine was measured in 205 pairs of capillary and venous plasma samples collected simultaneously at ≥24hr post dose in children, pregnant women and non-pregnant women receiving dihydroartemisinin-piperaquine as malaria chemoprevention. Standard three-dose regimen over three days applied to all participants with three 40mg dihydroartemisinin/320mg PQ tablets per dose for adults and weight-based dose for children. Correlation analysis was performed using the program Stata® SE12.1. Linear regression models were built using concentrations or logarithm transformed concentrations and the final models were selected based on maximal coefficient of determination (R2) and visual check.

Results

An LC-MS/MS method was developed and validated, utilizing methanol as a protein precipitation agent, a Gemini C18 column (50x2.0mm, 5μm) eluted with basic mobile phase solvents (ammonium hydroxide as the additive), and ESI+ as the ion source. This method had a calibration range of 10–1000 ng/mL and carryover was negligible. Correlation analysis revealed a linear relationship: Ccap = 1.04×Cven+4.20 (R2 = 0.832) without transformation of data, and lnCcap = 1.01×lnCven+0.0125, (R2 = 0.945) with natural logarithm transformation. The mean ratio (±SD) of Ccap/Cven was 1.13±0.42, and median (IQR) was 1.08 (0.917, 1.33).

Conclusions

Capillary and venous plasma PQ measures are nearly identical overall, but not readily exchangeable due to large variation. Further correlation study accounting for disposition phases may be necessary.

Introduction

Piperaquine (PQ) is a long-acting antimalarial drug currently used in combination with dihydroartemisinin for malaria treatment, and also studied for chemoprevention [1, 2]. PQ (Fig 1) is a weak base with pKa values around 8.6 and 6.5 and high lipophilicity (LogP = 6.2) at neutral and alkaline pH [3]. The free base form of PQ is poorly soluble in water, methanol (MeOH), and acetonitrile (MeCN), but very hydrophilic at low pH, and easily soluble in acidified solvents.

Fig 1.

Fig 1

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Chemical structures of piperaquine (left) and piperaquine-d6 (right).

Adsorption of PQ to a glass surface occurs if the solution is stored in a glass container, and loss of PQ signal is observed when a metal injection needle or metal tubing is used in an analytical system. Our group found over 50% PQ loss after storage in a glass tube overnight (S1 Table). The extent of adsorption is in the following order: silanized glass > glass > polypropylene plastic tube > Thermo Scientific™ low retention tubes. In plasma samples, over 97% of PQ binds to proteins [4, 5]. These properties present daunting challenges for method development. A considerable challenge is carryover between assays. Carryover could be due to the column (tailing peak), precolumn tubing, or autosampler. Numerous methods have been published for the quantification of PQ in plasma, including high performance liquid chromatography-ultraviolet/visible spectrophotometry (HPLC-UV) [5–7] and liquid chromatography tandem mass spectrometry (LC-MS/MS) [8–12]. Carryover was observed in nearly all LC-MS/MS methods. Here we report an LC-MS/MS method with negligible carryover and its application to venous and capillary samples simultaneously collected for correlation analysis.

Venous blood sampling via catheter remains to be the mainstream method for clinical sample collection. Capillary blood sample collection via finger or heel stick could be used as an alternative method, especially in rural areas and pediatric patients [2]. Current intensive pharmacokinetic designs are often relying on the merging of venous and capillary sampling [13, 14]. However, drug levels in capillary plasma may be different from those in venous plasma because of difference in matrix contents, such as oxygen and protein [15, 16]. Correlation between capillary and venous drug levels needs to be established in order to properly interpret and analyze results. A previous study has suggested capillary blood PQ concentrations are higher on average than venous blood PQ in malaria patients [17]. Here we report correlation of venous plasma and capillary plasma PQ concentrations in participants receiving dihydroartemisinin-PQ when enrolled in a malaria chemoprevention trial.

Methods

Materials

Piperaquine tetraphosphate tetrahydrate (MW 999.55, purity 99%) was purchased from A.K.Scientific Inc. (Union City, CA, USA). Piperaquine-d6 (PQ-d6, MW 541.55, isotopic purity ≥99%) was purchased from AlSAchim, SAS (IllKirch, France). Trichloroacetic acid (TCA, certified ACS reagent) and ammonium formate (NH4FA,certified ACS reagent), trifluoroacetic acid (TFA, Optima™ LC/MS grade), formic acid (FA, Optima™ LC/MS grade) and ammonium hydroxide (NH4OH) (Optima™ LC/MS grade), acetonitrile (MeCN, HPLC grade), methanol (MeOH, HPLC grade), and other common solvents (HPLC grade) were purchased from Fisher Scientific Co. (Fair Lawn, NJ, USA). Blank human plasma (K3EDTA added as anticoagulant) was obtained from Biological Specialty Co (Colmar, PA, USA).

Quantitation method

Sciex API2000 tandem mass spectrometer was coupled with a Perkin Elmer 200 series micro LC system. The LC column was Gemini C18 (50×2.0 mm, 5μm) fitted with a guard column (4×2.0 mm, 5μm) (Phenomenex Inc., Torrance, CA, USA), eluted with 10 mM NH4OH (A) and MeCN (B) in a gradient mode at a flow rate of 0.6 mL/min. ElectroSpray ionization in positive mode (ESI+) was used as the ion source with multiple reaction monitoring (MRM) of m/z 535/288 for PQ and m/z 541/294 for the IS (PQ-d6) for quantitation. PQ stock solution was prepared in MeCN-water (1:9, v/v) containing 0.5% formic acid. Calibration standard samples (10, 25, 50, 100, 250, 500, and 1000 ng/mL) and QC samples (30, 200, and 800 ng/mL) were prepared in blank plasma from two different stock solutions. Plasma samples (25 μL) were mixed with 25 μL 30 ng/mL PQ-d6 in MeCN-water (1:9, v/v) containing 0.5%FA, added 150 μL MeOH, briefly vortex-mixed, and centrifuged at 25,000g for 5min. Transferred ~100μL supernatant to plastic sample vials or 96-well plate in autosampler. Injection volume was 10 μL.

Validation

The method was validated based on the NIH-sponsored CPQA guidelines [18], which was adopted from the FDA guidelines [19]. A full validation includes precision and accuracy, matrix effect and recovery, dilution integrity and partial volume, stability, concomitant drug interference, and cross validation with a published method. Dilution integrity was evaluated by diluting the extra-high QC sample (3000 ng/mL) by 3-, 5-, and 10-fold with blank plasma. Partial volume was evaluated by mixing 12.5 μL QC samples with 12.5 μL blank plasma. Stability in plasma was evaluated at 4°C overnight (16hr) in glass vial and after 5 freeze-thaw cycles by comparing the treated samples with untreated samples in plastic microcentrifuge tubes. Long term stability in plasma at -70°C for 21 months was tested with the published method as the concentrations of old QC samples were made for the method [3]. To evaluate autosampler stability, the processed low and high QC samples were tested on the same day of processing (as control) and after 7 days in autosampler plastic vials. Stock solution stability was evaluated on UPLC-PDA system by diluting the stock to 10 ug/mL in MeCN-water (1:9) containing 0.5%FA. All measurements were performed in at least triplicates. Matrix effect was evaluated with 7 different lots of human plasma with K3EDTA as the anticoagulant. Set 1 samples were prepared by spiking both PQ and IS in 75% MeOH with final concentrations of 3.75, 25, and 100 ng/mL for PQ and 3.75 ng/mL for IS, corresponding to the final concentrations of PQ and IS from plasma samples after protein precipitation. Set 2 samples were spiked at the same concentration as Set 1 in extracted solutions from 7 lots of blank plasma, and Set 3 samples were prepared by spiking PQ in 7 lots of blank plasma with a final PQ concentration of 30, 200, and 800 ng/mL and then processing the plasma samples as described above.

Clinical sample analysis

The method applied to capillary versus venous plasma PQ correlation study, which is part of a pharmacokinetic study within the larger trials for malaria chemoprevention in pregnant women and children (ClinicalTrials.gov number, NCT02163447) [13, 14, 20, 21]. The study was conducted in Tororo, Uganda from December 2014 to May 2017. Eligible participants were pregnant women with ultrasound-estimated gestational age of 12–20 weeks and their children. Complete entry criteria were summarized previously [13, 14, 22]. The studies were approved by the Uganda National Council of Science and Technology and institutional review boards of Makerere University and the University of California, San Francisco. Written informed consent was obtained from adult study participants, and, for children, from their parents or guardians. The reported method was used to analyze 150 pairs of plasma samples from capillary and venous blood simultaneously collected 24 hr post last dose. We also modified a previously published method in our group to a lower calibration range of 0.5–50 ng/mL [3], and performed the required partial validation for this modification (S1 File). The modified method was used for 65 pairs of plasma samples from capillary and venous blood simultaneously collected from 7 to 84 days post 1st dose. Standard three-dose regimen over three days applied to all participants with three 40mg dihydroartemisinin/320mg PQ tablets per dose for adults [13] and weight-based dose for children [14].

Correlation analysis

Using STATA SE12.1, the relationship between capillary and venous plasma PQ concentrations was modelled using a linear relationship with estimated intercept and slope. The linear least squares regression models were built using concentrations or logarithm transformed concentrations and the final models were selected based on maximal coefficient of determination (R2) and visual check.

Results

1. Method development

The optimized MS parameters were shown in Table 1. Most published methods for analysis of PQ have utilized acidic mobile phase solvents. Alternatively, basic mobile phase solvents can be used. Lindegardh’s group published a method using 2.5 mM ammonium bicarbonate (pH10.0)-acetonitrile (15:85, v/v) as the mobile phase solvents [8]. Based on this method, we developed a new method on an API2000 system. A tailing peak was observed when we tried to use the same solvents [23], so we decided to use ammonium hydroxide in the mobile phase. Higher NH4OH concentration led to better peak shape, but lower signal intensity. Optimal peaks were observed using 10 mM NH4OH as mobile phase A, and 100% acetonitrile as mobile phase B (Fig 2). The final LC gradient program consists of 55% solvent B (0 min), from 55 to 100% B (0–1.5 min), 100% B (1.5–2.0 min), 100%-55% B (2.0–2.1 min), and 55% B (2.1–2.5 min). Surprisingly, no carryover peaks were observed with this method, though occasionally a small carryover peak was found in some runs, probably due to pH variation of mobile phase A. Ammonia may evaporate and carbon dioxide in air may dissolve in mobile phase A to change the pH over time. Therefore, we recommend that mobile phase A be prepared freshly every day before use. Disappearance of carryover peak may also possibly because the API2000 system is less sensitive.

Table 1. Optimized MS parameters.

Source parameters TEM,°C IS, v CAD, psi CUR, psi Gas1, psi Gas2, psi
500 2000 11 30 40 70
Compound parameters DP, v FP, v EP, v CE, v CEP, v CXP, v Dwell time, ms
535/288 (PQ) 76 360 12 49 20 10 250
541/294 (PQ-d6, I.S.) 76 360 12 49 20 10 250
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TEM, source temperature; IS, ionspray voltage; CUR, curtain gas, Gas1, nebulizer gas; Gas2, auxiliary gas; CAD, collision-activated dissociation; DP, declustering potential; FP, focusing potential; EP, entrance potential; CE, collision energy; CEP, collision cell entrance potential; CXP, collision cell exit potential.

Fig 2.

Fig 2

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Chromatograms of PQ representing blank plasma (dashed gray line), the LLOQ (solid black line) and blank plasma after ULOQ, indicating carryover (solid gray line).

2. Method validation

The lower limit of quantitation (LLOQ) for this assay was set at 10 ng/mL, with a S/N ratio = 9.7. The calibration range was 10–1000 ng/mL. The calibration curve was fitted with least square linear regression weighted by 1/x. The correlation coefficient (r) was typically > 0.9990. Representative chromatograms of PQ from blank plasma extract, LLOQ, and blank plasma extract injected after the upper limit of quantitation (ULOQ) are shown in Fig 2.

The intra-day precision (n = 6) over 3 days ranged from 1.2 to 4.0% at the three concentrations (30, 200, and 800 ng/mL), and inter-day precisions ranged from 2.7 to 4.6%, all of them within 15% deviation from mean values. The intra- and inter-day accuracy ranged from 0.4 to 9.1%and 5.1 to 5.3%, respectively, all within 15% deviation from the nominal values. At the LLOQ 10 ng/mL level, the precision and accuracy met the criteria of <20% (Table 2).

Table 2. Intra- and inter-day precision and accuracy.

 Nominal, Intra-day (n = 6) Inter-day (n = 18)
ng/mL 10.0 30.0 200 800 10.0 30.0 200 800
mean, ng/mL 9.97–10.2 30.1–32.7 202–217 828–853 10.1 31.5 211 842
RSD,% 4.8–8.3 2.4–4.0 2.2–3.6 1.2–3.3 6.2 4.6 4.2 2.7
%dev (-0.32)-2.3 0.4–9.1 0.8–8.5 3.5–6.6 0.6 5.1 5.5 5.3
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Absolute matrix effect was evaluated with mean peak areas from Set 1 (clean solution) and Set 2 (post-extraction spiked solution). A value of 100% indicated no matrix effect. If the value was >100%, ion enhancement was observed, and if <100%, ion suppression was observed. At low, medium, and high concentrations, the matrix effect for PQ was 107, 86.6, and 91.8%, respectively (Table 3). The IS normalized matrix effect (PQ/IS) was close to 100%. These results indicated that the matrix effect was not significant and compensated well by the deuterated IS.

Table 3. Matrix effect and recovery (n = 7).

 Conc PQ Peak Area, x104 IS Peak Area, x104 Matrix Effect Recovery
(ng/ml) Set 1 Set 2 Set 3 Set 1 Set 2 Set 3 PQ IS PQ/IS PQ IS
Low (30) 4.13±1.38 4.40±0.62 3.01±0.43 4.30±0.15 4.26±0.36 3.28±0.33 107 99.1 108 68.4 77.0
Med (200) 29.0±2.5 25.1±2.7 21.4±1.2 4.41±0.36 3.88±0.42 3.23±0.23 86.6 88.0 98.4 85.5 83.3
High (800) 110±3 101±6 878±7 4.15±0.12 3.66±0.35 3,24±0.29 91.8 88.2 104 87.3 88.6
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Set 1, MeOH-Water (3:1, v/v) solution; Set 2, post extraction spiked solution; Set 3, pre-extraction spiked solution.

The recovery of PQ at 30, 200, and 800 ng/mL was 68.4, 85.5, and 87.3% respectively. The mean recovery for the I.S. was 83.0%.

To determine dilution integrity, we diluted an extra high QC (3000 ng/mL) by 3-, 5-, and 10-fold. The accuracy (% deviation) for dilution samples ranged from -4.4 to 2.2 (S2 Table). We also tested partial volumes by taking half volume samples mixed with an equal volume of blank plasma. The % deviation for all partial volume samples ranged from 1.3 to 9.9 (S2 Table). These results confirm that plasma samples can be diluted by up to 10-fold.

Previously, we reported that PQ was stable in plasma at room temperature (21–24°C) for 9 days, at -70°C for 8 months, and after 3 freeze-thaw cycles [3]. In this report, we further tested and found that PQ is stable in plasma after 5 freeze-thaw cycles and at -70°C for at least 21 months. There was no difference in results for plasma samples prepared in glass versus plastic vials, likely due to PQ bound to plasma proteins. The processed samples were stable in autosampler plastic vials for at least 7 days, and stock solution was stable at -70°C for at least 3 years (Table 4). Noticeably, evaluation of stock solution stability with the API2000 yielded large variation from repeated injections; this is most likely due to the metal injection needle used for the API 2000 system. When switched to an Acquity UPLC-PDA system with a polyetheretherketone (PEEK) sample needle, reproducible results were obtained.

Table 4. Stability of PQ. Data represent mean (± SD).

Conditions Untreated*** treated % remained n
Plasma, glass container, 4°C, 16 hr
30 ng/mL 29.8±2.3 30.2±1.1 101 3
800 ng/mL 796±4 832±14 105 3
Autosampler plastic vials, 21–25°C, 7 days
30 ng/mL 32.8±1.7 29.4±1.1 89.6 4
800 ng/mL 865±27 857±19 99.1 4
Five freeze-thaw cycles
30 ng/mL 31.7±1.3 30.9±1.2 97.5 3
800 ng/mL 844±12 843±12 99.9 3
Plasma, -70°C, 21 months*
3 ng/mL 2.76±0.15 2.78±0.04 101 3
200 ng/mL 200±6 209±5 104 3
Stock, -70°C, 40 months**
  8170±605 8091±338 99 3
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* Measured with our previously published method

**measured with UPLC-PDA with peek tubing injection needle.

*** untreated samples were prepared fresh in plastic vials.

To test potential concomitant drug interference, lumefantrine, artemether, dihydroartemisinin, nevirapine, efavirenz, zidovudine, lamivudine, stavudine (D4T), lopinavir, nelfinavir, indinavir, saquinavir, and amprenavir were spiked into the medium QC samples. The differences of the spiked samples compared to the control medium QC were within 5%, confirming no interference from potential concomitant drugs (S3 Table).

To further validate the API2000 method, we re-analyzed 116 samples that were analyzed with the previously published method [3]. The concentration of those samples ranged from 9.06 to 553 ng/mL. Three samples were measured as below LLOQ. Among the 113 quantifiable samples, 100 samples (88.5%) were within 20% of the reference value (S4 Table).

3. Carryover

Having 6 nitrogen atoms with different pKa values, PQ can be found in multiple forms in solution. Due to this property, peak tailing and carryover peaks were often observed during the analysis of PQ. The majority of published LC-MS/MS methods utilized acidic mobile phase solvents [3, 9–12], while two methods used basic mobile phase solvents [8, 23] (Table 5). Carryover peaks were observed in nearly all published LC-MS/MS methods. Most methods maintained the carryover peaks within 20% LLOQ. In the method published by Hodel et al, the carryover peak of PQ was >100% LLOQ [10], partially due to the wide calibration range. Recently, our group published a method on an API 5000 LC-MS/MS system [3] in which carryover from the column was removed, but carryover from the autosampler/injection was still present at ~0.08% of ULOQ (250 ng/mL). Accordingly, the LLOQ was raised to 1.5 ng/mL. In the modified method with a calibration range of 0.5-50ng/mL, the residual carryover peak was maintained within 20% LLOQ (S3 File). Our group also developed a method using a basic mobile phase [10], based on a method published by Lindegardh et al [2]. The peak tailing and carryover were significant (S3 File). In the current report, we changed the basic additive from 2.5 mM NH4HCO3 to 10 mM NH4OH, and changed the column from Zorbax Eclipse C18 (50x2.1mm, 5μm) to Gemini C18 (50x2.0, 5μm). With these changes, the carryover peak was reduced markedly. Although carryover peak was observed in some runs, in most cases no carryover peaks were observed with this method (S4 File). It might possibly be due to the less sensitivity of the instrument and/or the narrow calibration range spanning only 100-fold when compared to other methods. It is also likely due to the basic mobile phase condition. We speculate that the ionic PQ molecule causes the carryover, which could diminish at basic condition where PQ is uncharged. Considering PQ pka = 8.6, PQ will be uncharged if mobile phase pH >10.6, and analytical columns applicable at pH 11 or higher are now available with the advancement of column technology.

Table 5. Published methods for PQ quantification.

Reference Instrument Column Mobile phase Sample preparation Calibration range Carryover Retention factor k
Hodel et al, 2009 TSQ Quantum (ESI+) Atlantis dC18 (50x2.1mm) 20mM NH4FA 0.5% FA; MeCN 0.5%FA PPT: 200uL plasma+700uL MeCN 2–4000 ng/mL yes (>100% LLOQ) 9.7
Singhal et al, 2007 API4000 Q-trap (ESI+) Chromolith SpeedROD RP-18e (50x4.6mm) NH4AC-MeOH-FA-NH3 PPT: 50uL plasma+300uL MeOH 1–250 ng/mL yes (not specified) 0.6
Lindegardh, et al, 2008 API5000 (ESI+) Gemini C18 (50x2.0mm) NH4HCO3-MeCN SPE: 50 uL plasma 1.5–500 ng/mL <15%LLOQ 2.4
Lee et al, 2011 API2000 (ESI+) Zorbax C18 (50x2.1mm, 5um) NH4HCO3-MeCN Dilution: 50uL PBS samples +IS 20-1000ng/mL yes (18–125%LLOQ) 4
Kjellin, et al, 2014 API5000 (APCI+) Pursuit PFP (50x2.0mm, 3um)) NH4FA-TFA-MeCN PPT: 25uL plasma+100uL MeOH-TCA 1.5-250ng/mL <20%LLOQ 4
Liu et al, 2017 API5500 Q-trap (ESI+) Venusil XBP-C18 (50x2.1mm, 5um) 2mMNH4AC 0.15%FA 0.05%TFA-MeCN PPT: 40uL plasma+HCl+160 uL MeCN 2–400 ng/mL ≤LLOQ 3
Aziz et al, 2017 API4000 (ESI+) Express C18 (30x2.1mm, 5um) 0.1%FA in Water-MeCN PPT: 100uL plasma+ 300uL 1%FA in water 3.9–2508 nM Yes (not specified) 29
This report API5000 (APCI+) Pursuit PFP (50x2.0mm, 3um) NH4FA-TFA-MeCN PPT: 25uL plasma+100uL MeOH-TCA 0.5-50ng/mL <20%LLOQ 4
This report API2000 (ESI+) Gemini C18 (50x2.0mm,5um) 10mMNH4OH-MeCN PPT: 25uL plasma+25ul IS+150uL MeOH 10–1000 ng/mL no 6
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4. Analysis of clinical samples

A total of 215 pairs of plasma samples simultaneously collected from capillary and venous blood were analyzed. Five pairs of samples were below LLOQ (0.5ng/mL) for both capillary and venous PQ, 4 capillary samples were below LLOQ with measurable venous PQ, and 1 venous sample was below LLOQ with measurable capillary PQ, yielding a total of 205 pairs of quantifiable data points for correlation analysis.

5. Correlation of PQ in capillary versus venous plasma

A total of 205 pairs of data above LLOQ were obtained from samples collected from 24 hr to 82 days post last dose in children, pregnant women and non-pregnant adults. Simple linear regression yields an equation: Ccap = 1.04×Cven + 4.20, R2 = 0.832 (Table 6). The 95% confidence intervals (CI) of the intercept included zero (-0.878, 9.27), and the p-value was 0.105, suggesting the difference from zero was not statistically significant. The slope was 1.04 with 95% CI (0.978, 1.11). The results suggest PQ concentrations in capillary and venous plasma appear to be correlated in a simple linear relationship. However, large variation led to a scattered correlation plot (Fig 3), a significant portion of samples could not be explained by the linear equation, making extrapolation of PQ concentrations from capillary to venous plasma complicate. With natural log-transformed data, improved correlation was obtained with R2 values of 0.945 and the equation lnCcap = 1.01×lnCven + 0.013. In general, slightly higher PQ concentrations were found in capillary samples, with a median value of 56.3 ng/mL versus 50.4 ng/mL in venous samples (p<0.0001). The mean ratio (±SD) of Ccap/Cven was 1.13±0.42, and median with interquartile range (IQR) was 1.08 (0.917, 1.33). However, 78 of 205 capillary PQ values (38%) were lower than corresponding venous PQ values.

Table 6. Correlation of capillary and venous PQ plasma concentrations.

  Total (n = 205) 24 hr post last dose (n = 150) ≥ 7 days post dose (n = 55)
Cven, ng/mL 50.4 (0.504, 251) 70.8 (9.67, 251) 3.41 (0.504, 20.4)
Ccap, ng/mL 56.3 (0.584, 292) 73.5 (16.7, 292) 2.84 (0.584, 22.6)
P value <0.0001 <0.0001 0.38
Correlation equition: Ccap = a × Cven + b
a 1.04 (0.978, 1.11) 0.984 (0.886, 1.08) 0.965 (0.828, 1.10)
b 4.20 (-0.878, 9.27) 10.6 (1.68, 19.5) -0.182 (-1.02, 0.661)
R2 0.832 0.729 0.789
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Concentrations represent medians (range) and correlation parameters represent means (95%CI).

Fig 3. Linear regression of capillary versus venous plasma PQ.

Fig 3

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Considering the impact of pharmacokinetic stages, we performed sub-analysis with data from 24 hr post last dose and ≥ 7 days post dose. At 24 hr post last dose, we collected 150 pairs of data, 57 from pregnant women, 63 from children under 2 years old, and 30 from non-pregnant women. The correlation equation at this time interval in each population was published previously with natural log-transformed data, which yielded better correlation than non-transformed data [13, 14]. When all data at 24 hr post last dose were combined (n = 150), simple linear regression yielded a coefficient of determination (R2) of 0.729 (Table 6). We observed a slightly higher capillary PQ concentration on average, with the median value of 73.5 ng/mL versus 70.8 ng/mL for venous PQ (p<0.0001). The mean ratio (±SD) of Ccap/Cven was 1.17±0.37, and median (IQR) was 1.12 (0.952, 1.34). The data suggest that the capillary concentration is slightly higher at the distribution phase. However, 48 of the 150 capillary PQ concentrations (32%) were lower than venous PQ concentrations. Natural log-transformed data did not improve the correlation. At ≥ 7 days post dose, we collected 55 pairs of data from children, yielding a linear regression equation Ccap = 0.965×Cven− 0.182 and R2 = 0.789. Log-transformed data did not improve the correlation. The median PQ concentrations for venous and capillary plasma were 3.41ng/mL and 2.84ng/mL, respectively, and the difference was not statistically significant (p = 0.38). The mean ratio (±SD) of Ccap/Cven was 1.03±0.52, and median (IQR) ratio was 0.974 (0.793, 1.21). Over 50% of capillary PQ concentrations were lower than venous PQ concentrations, suggesting that at the terminal elimination phase venous PQ tends to be higher than capillary PQ. A study in Burkina Faso reported day 7 PQ concentrations in capillary plasma were higher than those in venous plasma with median (range) concentration at 67 (49–84) in capillary versus 41 (27–59) in venous plasma (n = 186, p<0.001) [24]. Our result is different, likely because 54 of the 55 pairs of samples were collected beyond 20 days after dose, which are better representatives of the terminal elimination phase.

A study in Thailand estimated correlation of dihydroartemisinin-piperaquine administration on malaria patients aged >2 years [17]. Venous blood PQ were nearly always higher than venous plasma PQ with a median (90% range) ratio of 2.15 (0.91, 5.26), suggesting PQ concentrated in red blood cells; while the difference between capillary blood and venous blood PQ concentrations was smaller with median (90% range) ratio of 1.66 (0.92, 3.03). After day 3 when parasitaemia had cleared, a simple relationship was found: the venous blood PQ concentration = (capillary blood PQ concentration)0.9. However, similar to what we observed here, there were also large variations in that study, leading to the conclusion that measurements of venous and capillary PQ concentration are not readily interchangeable. In contrast, another antimalarial, lumefantrine, showed good linear correlation at a 1:1 ratio between capillary and venous samples, with coefficient of determination R2>0.95 [21]. This is probably due to the complex pharmacokinetic profile of PQ, such as multiple peak concentrations, slow distribution of PQ requiring longer time to reach equilibrium, large distribution volume (up to 874L/kg), and long elimination half-life (up to 28 days) [25]. It is likely not feasible to find a linear correlation during early pharmacokinetic phases, as equilibrium between different compartments is not reached and varied among individuals. Correlation may be better accessed during the steady state and the elimination phase., e.g day 14 or 28.

Conclusions

We reported a method for quantitation of PQ in plasma with a calibration range of 10–1000 ng/mL. Carryover was negligible in the method.

The concentrations of PQ in capillary and venous plasma were correlated in a linear relationship. However, due to large variations, exchange of PQ concentrations between capillary and venous plasma will compromise precision of the results. Correlation study accounting for disposition phases may be necessary.

Supporting information

S1 Table. Adsorption of PQ on container surface.

(XLSX)

Click here for additional data file. (12KB, xlsx)
S2 Table. Dilution and partial volume precision and accuracy for PQ.

(XLSX)

Click here for additional data file. (11.8KB, xlsx)
S3 Table. Interference of potential concomitant drugs.

(XLSX)

Click here for additional data file. (12.7KB, xlsx)
S4 Table. Cross validation using the previous API5000-based method as reference.

(XLSX)

Click here for additional data file. (16.8KB, xlsx)
S1 File. Partial validation of the modified PQ assay.

(PDF)

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S2 File

(DTA)

Click here for additional data file. (11.6KB, dta)
S3 File

(PDF)

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S4 File

(PDF)

Click here for additional data file. (198.6KB, pdf)

Acknowledgments

We wish to thank the volunteers participating in this study, the study coordinators at the participating sites, and supporting staff at Drug Research Unit at UCSF.

Data Availability

All relevant data are within the paper and its Supporting Information files.

Funding Statement

This work is supported by the National Institutes of Health (NIH), grant numbers R01AI117001 (PJR, FA), 4P01HD059454-09 (GD), and P30AI027763 (FA). https://www.nih.gov/grants-funding. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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Decision Letter 0

Henk D F H Schallig

10 Mar 2020

PONE-D-20-05889

Determination of piperaquine concentration in human plasma and the correlation of capillary versus venous plasma concentrations

PLOS ONE

Dear Dr Huang,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has some merit but does not meet PLOS ONE’s publication criteria as it currently stands.  In particular, the expert reviewer has major concerns about the fact that the authors cannot justify the absence of carry-over in their method by showing additional results (see reviewer's requests in the comments to the author),  Consequently the statements and conclusions in the manuscript are not sufficiently supported by the results. If this can not be appropriately addressed in a revision we will move to rejection of the manuscript. We now invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

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Reviewer #1: The authors describe a novel chromatography method to separate and eventually quantify piperaquine in human plasma, plus an additional comparison of venous plasma concentrations versus capillary plasma concentrations. Regarding the detection method, at least 5 other piperquine methods have been described in the literature, and also the comparison of venous versus capillary concentrations has extensively been described, I believe one of the first papers was in 2010 from Lindegardh's group.

The authors propose that the method described here is the first method that does not exhibit carry-over, a characteristic which is present for piperaquine given its high adsorbance to a variety of surfaces. However, the authors appear to describe two methods in the current paper, a novel one with a new chromatography and a range of 10-1000 ng/mL, and an older one, previously published in 2014, with a range of 0.5-50 ng/mL. This is actually very unclear in the manuscript, LLOQ's are constantly being mixed up and also the methodology only appears to describe the method of 10-1000 ng/mL, while little details are given regarding the method of 0.5-50 ng/mL. I have a few major queries and comments:

(1) The main point of the new method (10-1000 ng/mL) appears to be the avoidance of carry-over, apparently achieved according to the authors, by the new chromatography. Carry-over can be easily avoided by injecting blank samples after a high concentrations. The only benefit of avoiding carry-over would be that the order of measuring samples does not need to be accounted for, which would be an added benefit. However, the another second method was used by the authors, using a more sensitive machine (API5000 instead of API2000) to measure the low concentrations, which was actually previously published already (but then with an LLOQ of 1.5 ng/mL). Th application of this second method for concentrations in the lowest range still requires the analyst to know which samples contain which concentrations, to know which samples should be measured at which machine. This means that all lower concentrations are not even measured with the method, in the same assay, as any of the higher concentrations, and thus the added value of having no carry-over in the method with the 10-1000 ng/mL is, in my opinion, absent.

(2) Perhaps more importantly, I have the impression that the absence of noticing a carry-over with the new method (10-1000 ng/mL) is an artefact of the methodology used and the high limit of quantification of this method. Actually, as the authors showed in their previous publication on piperquine LCMSMS analysis from 2014 in Bioanalysis, the carry-over after an ULOQ using the method that they used for quantifying the concentration range of 0.5-50 ng/mL, was only 0.2 ng/mL. This means that the LLOQ of the new method (10 ng/mL) is about 50 times the signal of the carry-over. Given that the API2000 is a much less sensitive machine and the high LLOQ with this method, I simply assume that a carry-over of maximally 0.2 ng/mL is simply not visible with this unsensitive methodology. I would be happy to see my statements corrected by any additional evidence provided by the authors, but based on what currently is provided the statement that carry-over is absent with the new chromatography is not supported by the results.

(3) More details are needed regarding the origin and quality of chemicals. Moreover, I suggest the authors to adhere to the international bioanalytical validation requirements as issued by FDA and/or EMA.

(4) The ULOQ seems to be high for the encountered concentrations in the clinical samples (maximally 250-300 ng/mL), why and how was chosen for this calibration range?

(5) Differences between venous and capillary plasma concentrations have previously been published, however these are not well discussed in the current discussion.

(6) The authors suggest a linear relationship between venous and capillary plasma concentrations. The fit of this relationship appears to be far from optimal and extrapolation of venous concentrations from capillary concentrations appears difficult, the manuscript would benefit if the authors could acknowledge this limitation and discuss its impact.

(7) Regarding this linear relationship between venous and capillary concentrations: the authors state in the discussion: "This is probably due to the complex PK profile of PQ, such as multiple peak concentrations and slow distribution of PQ requiring longer time to reach equilibrium." If this would be the case a linear relationship over the whole concentration range seems physiologically not correct, the manuscript would benefit if the authors could consider and discuss this.

(8) Ethics: the registration numbers relating to approval by any of the ethical/institutional review boards should preferably be included in the manuscript for full ethical transparency.

**********

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Reviewer #1: No

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PLoS One. 2020 May 29;15(5):e0233893. doi: 10.1371/journal.pone.0233893.r002

Author response to Decision Letter 0

24 Apr 2020

Editor's comments:

1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at http://www.plosone.org/attachments/PLOSOne_formatting_sample_main_body.pdf and http://www.plosone.org/attachments/PLOSOne_formatting_sample_title_authors_affiliations.pdf

Response: It was written referring to the templates

2. In your Methods section, please provide additional information about the participant recruitment method. Please ensure you have provided sufficient details to replicate the analyses such as: a) the recruitment date range (month and year), b) a description of any inclusion/exclusion criteria that were applied to participant recruitment, and c) a description of how participants were recruited.

Response: The information for clinical trial was published previously and can be found in the reference. The following sentences are added now: “(ClinicalTrials.gov number,NCT02163447) [13, 14, 19, 20]. The study was conducted in Tororo, Uganda from December 2014 to May 2017. Eligible participants were pregnant women with ultrasound-estimated gestational age of 12-20 weeks and their children. Complete entry criteria were summarized previously[13, 14, 21].”

3. We note that you have indicated that data from this study are available upon request. PLOS only allows data to be available upon request if there are legal or ethical restrictions on sharing data publicly. For more information on unacceptable data access restrictions, please see http://journals.plos.org/plosone/s/data-availability#loc-unacceptable-data-access-restrictions

Response: the concentration data for the correlation analysis is now provided in the supplemental file (S2 file.dta).

Reviewer #1: The authors describe a novel chromatography method to separate and eventually quantify piperaquine in human plasma, plus an additional comparison of venous plasma concentrations versus capillary plasma concentrations. Regarding the detection method, at least 5 other piperquine methods have been described in the literature, and also the comparison of venous versus capillary concentrations has extensively been described, I believe one of the first papers was in 2010 from Lindegardh's group.

The authors propose that the method described here is the first method that does not exhibit carry-over, a characteristic which is present for piperaquine given its high adsorbance to a variety of surfaces. However, the authors appear to describe two methods in the current paper, a novel one with a new chromatography and a range of 10-1000 ng/mL, and an older one, previously published in 2014, with a range of 0.5-50 ng/mL. This is actually very unclear in the manuscript, LLOQ's are constantly being mixed up and also the methodology only appears to describe the method of 10-1000 ng/mL, while little details are given regarding the method of 0.5-50 ng/mL. I have a few major queries and comments:

Response: Thank you for your careful review. The focus of this manuscript is on the new method. The modification of the older method with a lower LLOQ (0.5 ng/mL) was only briefly described but validation data can be found in supporting information (S1 file). Method details can be found in the older method published in Bioanalysis.

(1) The main point of the new method (10-1000 ng/mL) appears to be the avoidance of carry-over, apparently achieved according to the authors, by the new chromatography. Carry-over can be easily avoided by injecting blank samples after a high concentrations. The only benefit of avoiding carry-over would be that the order of measuring samples does not need to be accounted for, which would be an added benefit. However, the another second method was used by the authors, using a more sensitive machine (API5000 instead of API2000) to measure the low concentrations, which was actually previously published already (but then with an LLOQ of 1.5 ng/mL). The application of this second method for concentrations in the lowest range still requires the analyst to know which samples contain which concentrations, to know which samples should be measured at which machine. This means that all lower concentrations are not even measured with the method, in the same assay, as any of the higher concentrations, and thus the added value of having no carry-over in the method with the 10-1000 ng/mL is, in my opinion, absent.

Response: I agree that impact of carryover can be avoided by injecting blank samples after a higher concentration. However, this is inconvenient for unknown samples, especially if we submit an overnight run, reinjections are required on the following day. The new method (10-1000ng/mL) was developed to support intensive PK studies with expected Cmax up to 1000 ng/mL. We analyzed ~2000 intensive PK samples with this new method. Only samples at elimination tail end were analyzed with the modified older method. There are 22% (455 out of 2075) samples above 250ng/mL, the ULOQ of our older published method. By avoiding carryover and dilution, we minimize reanalysis to save samples and efforts. This is a significant benefit, especially for plasma samples collected from pediatric participants, the sample volume is very limited.

(2) Perhaps more importantly, I have the impression that the absence of noticing a carry-over with the new method (10-1000 ng/mL) is an artefact of the methodology used and the high limit of quantification of this method. Actually, as the authors showed in their previous publication on piperquine LCMSMS analysis from 2014 in Bioanalysis, the carry-over after an ULOQ using the method that they used for quantifying the concentration range of 0.5-50 ng/mL, was only 0.2 ng/mL. This means that the LLOQ of the new method (10 ng/mL) is about 50 times the signal of the carry-over. Given that the API2000 is a much less sensitive machine and the high LLOQ with this method, I simply assume that a carry-over of maximally 0.2 ng/mL is simply not visible with this unsensitive methodology. I would be happy to see my statements corrected by any additional evidence provided by the authors, but based on what currently is provided the statement that carry-over is absent with the new chromatography is not supported by the results.

Response: Thanks for the thoughtful comments. The API2000 is indeed 20-100 fold less sensitive than API5000 depending on types of compounds. But please keep in mind, carryover amount is associated with the ULOQ. The carryover of 0.2ng/mL is based on the ULOQ of 250ng/mL and lower carryover was observed with a ULOQ of 50 ng/mL (Supporting S3 file). I project a carryover of ~0.8ng/mL following injection of a 1000ng/mL samples with the older method. Indeed, we observed significant carryover in an earlier in vitro study with API 2000 system using a calibration range of 20-1000ng/mL (Supporting S3 file). We also observed carryover sometimes in the new method (See supporting materials S4 file, page 1), but in most cases, there is no carryover (Supporting S4 file). Whereas, we did notice the sensitivity of the API2000 varied in different days.

(3) More details are needed regarding the origin and quality of chemicals. Moreover, I suggest the authors to adhere to the international bioanalytical validation requirements as issued by FDA and/or EMA.

Response: Sentences were reworded to clarify the quality of chemicals (line 88-91). We validated methods based on CPQA guidelines, which is based on the FDA guidelines, but with more restrictions. For example, Stock solution used for QC samples needs to a separately prepared solution from the stock solution used for calibrators. In FDA guidelines, this is not required as long as the stock solution was verified to be accurate.

(4) The ULOQ seems to be high for the encountered concentrations in the clinical samples (maximally 250-300 ng/mL), why and how was chosen for this calibration range?

Response: please see response to question #1.

(5) Differences between venous and capillary plasma concentrations have previously been published, however these are not well discussed in the current discussion.

Response: discussion and citation are added now. The following sentences were added in line 309 to 314: “A study in Burkina Faso reported day 7 PQ concentrations in capillary plasma were higher than those in venous plasma with median (range) concentration at 67 (49-84) in capillary versus 41 (27-59) in venous plasma (n=186, p<0.001). Our result is different, likely because 54 of the 55 pairs of samples were collected beyond 20 days after dose, which are better representatives of the terminal elimination phase.”

One additional reference was found and cited: Zongo I, Some FA, Somda SA, Parikh S, Rouamba N, Rosenthal PJ, et al. Efficacy and day 7 plasma piperaquine concentrations in African children treated for uncomplicated malaria with dihydroartemisinin-piperaquine. PLoS One. 2014;9(8):e103200. doi: 10.1371/journal.pone.0103200. PubMed PMID: 25133389; PubMed Central PMCID: PMCPMC4136730.

(6) The authors suggest a linear relationship between venous and capillary plasma concentrations. The fit of this relationship appears to be far from optimal and extrapolation of venous concentrations from capillary concentrations appears difficult, the manuscript would benefit if the authors could acknowledge this limitation and discuss its impact.

Response: thanks for the suggestion. We write additional discussion now in Line 273-276 as follows: “ The results suggest PQ concentration in capillary and venous plasma is likely at 1:1 ratio. However, Large interindividual variation led to a scattered correlation plot (Figure 3), a significant portion of samples are not at 1:1 ratio, making extrapolation of PQ concentration from capillary to venous plasma difficult.”

(7) Regarding this linear relationship between venous and capillary concentrations: the authors state in the discussion: "This is probably due to the complex PK profile of PQ, such as multiple peak concentrations and slow distribution of PQ requiring longer time to reach equilibrium." If this would be the case a linear relationship over the whole concentration range seems physiologically not correct, the manuscript would benefit if the authors could consider and discuss this.

Response: Thanks for the comments, additional discussion is added to reflect our opinion that correlation may be better at the late elimination phase. The following sentences were added in line 329-333:” It is likely not feasible to find a good correlation during early pharmacokinetic phases, as equilibrium between different compartments is not reached and varied among individuals. Better correlation may be obtained if correlation was performed at single time points in the elimination phase., e.g day 14 or 28.”

(8) Ethics: the registration numbers relating to approval by any of the ethical/institutional review boards should preferably be included in the manuscript for full ethical transparency.

Response: The clinical trials.gov registration number is provided (NCT02163447).

Decision Letter 1

Henk D F H Schallig

30 Apr 2020

PONE-D-20-05889R1

Determination of piperaquine concentration in human plasma and the correlation of capillary versus venous plasma concentrations

PLOS ONE

Dear Dr Huang,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

The expert reviewer has assessed your revised manuscript. Unfortunately, you did not address or meet all the required revisions requested by the reviewer. You have now the oportunity to correct this further. We want to receive a clear response to the issues raised by the reviewer and you must address all points raised.

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Reviewer #1: (No Response)

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Reviewer #1: Yes

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3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

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Reviewer #1: Yes

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Reviewer #1: Yes

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6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: I thank the authors for their revisions. I have a few more comments, the numbering below refers to my original comments and the authors' replies to these points:

(2) I believe the authors acknowledge my observation regarding the lower sensitivity of the equipment which is probably the reason for not observing the carry-over, instead of the adjusted chromatography. The authors suggest that carry-over would amount to 0.8 ng/mL for a 1000 ng/mL sample, this would indeed mean that carry-over remains undetected with the method with LLOQ of 10 ng/mL, but nevertheless might still be present. This needs to be clearly acknowledged in the manuscript as the authors still suggest now throughout the manuscript that carry-over is avoided due to the new chromatography method, a statement which I do not see supported by any data.

(6) This statement regarding the ratio between plasma and venous samples would benefit from mentioning some quantification about the variability, e.g. an easy way to illustrate the level of variability would be mentioning the range/IQR of the quantified ratio's for each paired sample set. Please also remove the word 'interindividual' as the variability is not due to variability between patients, but rather between sample time points (at least this is what the authors suggest).

(7) I assume the authors intended here 'linear correlation' and not just 'correlation'. Additionally, correlation might be better assessed during steady-state of the pharmacokinetics.

**********

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PLoS One. 2020 May 29;15(5):e0233893. doi: 10.1371/journal.pone.0233893.r004

Author response to Decision Letter 1

13 May 2020

Reviewer #1:

(2) I believe the authors acknowledge my observation regarding the lower sensitivity of the equipment which is probably the reason for not observing the carry-over, instead of the adjusted chromatography. The authors suggest that carry-over would amount to 0.8 ng/mL for a 1000 ng/mL sample, this would indeed mean that carry-over remains undetected with the method with LLOQ of 10 ng/mL, but nevertheless might still be present. This needs to be clearly acknowledged in the manuscript as the authors still suggest now throughout the manuscript that carry-over is avoided due to the new chromatography method, a statement which I do not see supported by any data.

Response: We want to keep the explanation open to multiple posibilities. We observed carryover peak during early validation, but in later experiments and routine sample analysis the carryover peak diminished, we don’t know the exact reasons for this improvement. I acknowledge the possibility that reviewer brought up and now add the following sentences:

Line 176-177: Disappearance of carryover peak may also possibly because the API2000 system is less sensitive.

Line 260-260: It might possibly be due to the less sensitivity of the instrument and/or the narrow calibration range spanning only 100-fold when compared to other methods. It is also likely due to the basic mobile phase. We speculate that the ionic PQ molecule causes the carryover, which could diminish at basic condition where PQ is uncharged. Considering PQ pka=8.6, PQ will be uncharged if mobile phase pH >10.6, and analytical columns applicable at pH 11 or higher are now available with the advancement of column technology.

(6) This statement regarding the ratio between plasma and venous samples would benefit from mentioning some quantification about the variability, e.g. an easy way to illustrate the level of variability would be mentioning the range/IQR of the quantified ratio's for each paired sample set. Please also remove the word 'interindividual' as the variability is not due to variability between patients, but rather between sample time points (at least this is what the authors suggest).

Response: The mean and median ration with IQR are described later in the paragraph. I reworded the sentences slightly as follows:

Line 278-287“ The results suggest PQ concentrations in capillary and venous plasma appear to be correlated in a simple linear relationship. However, large variation led to a scattered correlation plot (Figure 3), a significant portion of samples could not be explained by the linear equation, making extrapolation of PQ concentrations from capillary to venous plasma complicate……. The mean ratio (±SD) of Ccap/Cven was 1.13±0.42, and median with interquartile range (IQR) was 1.08 (0.917, 1.33). However, 78 of 205 capillary PQ values (38%) were lower than corresponding venous PQ values.”

Line 354-356: due to large variations, exchange of PQ concentrations between capillary and venous plasma will compromise precision of the results.

(7) I assume the authors intended here 'linear correlation' and not just 'correlation'. Additionally, correlation might be better assessed during steady-state of the pharmacokinetics.

Response: Thanks for your constructive suggestion. The sentences are updated as follows.

It is likely not feasible to find a linear correlation during early pharmacokinetic phases,….. . Correlation may be better accessed during the steady state and the elimination phase…

Decision Letter 2

Henk D F H Schallig

15 May 2020

Determination of piperaquine concentration in human plasma and the correlation of capillary versus venous plasma concentrations

PONE-D-20-05889R2

Dear Dr. Huang,

We are pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it complies with all outstanding technical requirements.

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With kind regards,

Henk D. F. H. Schallig, Ph.D

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: All comments have been addressed

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

**********

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: (No Response)

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Acceptance letter

Henk D F H Schallig

21 May 2020

PONE-D-20-05889R2

Determination of piperaquine concentration in human plasma and the correlation of capillary versus venous plasma concentrations

Dear Dr. Huang:

I am pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

If your institution or institutions have a press office, please notify them about your upcoming paper at this point, to enable them to help maximize its impact. If they will be preparing press materials for this manuscript, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org.

For any other questions or concerns, please email plosone@plos.org.

Thank you for submitting your work to PLOS ONE.

With kind regards,

PLOS ONE Editorial Office Staff

on behalf of

Dr. Henk D. F. H. Schallig

Academic Editor

PLOS ONE

Associated Data

    This section collects any data citations, data availability statements, or supplementary materials included in this article.

    Supplementary Materials

    S1 Table. Adsorption of PQ on container surface.

    (XLSX)

    Click here for additional data file. (12KB, xlsx)
    S2 Table. Dilution and partial volume precision and accuracy for PQ.

    (XLSX)

    Click here for additional data file. (11.8KB, xlsx)
    S3 Table. Interference of potential concomitant drugs.

    (XLSX)

    Click here for additional data file. (12.7KB, xlsx)
    S4 Table. Cross validation using the previous API5000-based method as reference.

    (XLSX)

    Click here for additional data file. (16.8KB, xlsx)
    S1 File. Partial validation of the modified PQ assay.

    (PDF)

    Click here for additional data file. (359.2KB, pdf)
    S2 File

    (DTA)

    Click here for additional data file. (11.6KB, dta)
    S3 File

    (PDF)

    Click here for additional data file. (647.6KB, pdf)
    S4 File

    (PDF)

    Click here for additional data file. (198.6KB, pdf)

    Data Availability Statement

    All relevant data are within the paper and its Supporting Information files.

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