key: cord-0822468-8oiu3m6n
authors: Bodur, Süleyman; Erarpat, Sezin; Günkara, Ömer Tahir; Bakırdere, Sezgin
title: Accurate and sensitive determination of hydroxychloroquine sulfate used on COVID-19 patients in human urine, serum and saliva samples by GC-MS
date: 2021-01-30
journal: J Pharm Anal
DOI: 10.1016/j.jpha.2021.01.006
sha: a25a5c30c6fb09e47bab1538f44064226280a144
doc_id: 822468
cord_uid: 8oiu3m6n

A rapid, accurate, and sensitive analytical method, ultrasonication-assisted spraying based fine droplet formation–liquid phase microextraction–gas chromatography–mass spectrometry (UA-SFDF-LPME-GC-MS), was proposed for the determination of trace amounts of hydroxychloroquine sulfate in human serum, urine, and saliva samples. To determine the best extraction strategy, several liquid and solid phase extraction methods were investigated for their efficiencies in isolation and preconcentration of hydroxychloroquine sulfate from biological matrices. The UA-SFDF-LPME method was determined to be the best extraction method as it was operationally simple and provided accurate results. Variables such as the extraction solvent, spraying number, sodium hydroxide concentration and volume, sample volume, mixing method, and mixing period were optimized for the proposed method using the one-variable-at-a-time approach. In addition, Tukey’s method based on a post hoc comparison test was employed to evaluate the significant difference between the parameters inspected. After the optimization studies, the limit of detection (LOD) and limit of quantification (LOQ) were determined to be 0.7 and 2.4 μg/kg, respectively. The sensitivity of the GC-MS system based on the LOD was enhanced approximately 440-fold when the UA-SFDF-LPME method was employed. Spiking experiments were also conducted for the human serum, urine, and saliva samples to determine the applicability and accuracy of the proposed method. Recoveries for the human serum, urine, and saliva samples were found to be in the ranges 94%–102%, 95%–105%, and 93%–102%, respectively. These results were satisfactory and indicated that the hydroxychloroquine sulfate level in the above biological samples could be analyzed using the proposed method.

. Chloroquine and hydroxychloroquine are widely 50 used as drug active compounds for the treatment of malaria [3] . Hydroxychloroquine is the 51 hydroxyl derivative of chloroquine that is synthesized to impart higher water solubility, lower 52 toxicity, and fewer side effects than chloroquine [4] . Literature survey on the effect of 53 hydroxychloroquine on coronavirus suggests that it can inhibit the virus replication process 54 and its fusion to the cell membrane [5, 6] . Unfortunately, hydroxychloroquine has side effects 55 such as vomiting, diarrhea, and gastrointestinal diseases. The more serious side effects include 56 retinopathy and QTc interval prolongation, which can cause ventricular arrhythmias and 57 sudden cardiac death [5] . It has been also reported that hydroxychloroquine usage causes 58 itching, intravascular hemolysis, rashes, and bone marrow suppression [7] . Therefore, an 59 accurate, sensitive, and rapid analytical method is required for the determination of 60 hydroxychloroquine in biological samples to scientifically evaluate its possible effects on 61 humans. 62 Generally, hydroxychloroquine is qualified and quantified by hyphenated and 63 electroanalytical techniques such as high performance liquid chromatography-ultraviolet 64 detection (HPLC-UV) [8] , high performance liquid chromatography-fluorescence detection preconcentrate the analyte prior to GC-MS measurements. Parameters of the SFDF-LPME 96 method were optimized by the one-variable-at-a-time approach. Tukey's method based on a 97 post hoc comparison test was utilized to evaluate the significant difference in pair-wise 98 comparisons and determine the optimum extraction conditions. After determining the limit of 99 detection (LOD), limit of quantification (LOQ), and linear range for the developed method, with an Agilent 5973 mass spectrometry detector (USA). The chromatographic separation 118 was carried out on an HP5MS column with the following dimensions: 30 m length, 250 µm inner diameter, and 0.25 µm film thickness. Helium was used as the mobile phase at a flow 120 rate of 2.8 mL/min. Inlet temperature and injection volume were set at 290 °C and 1.0 µL, 121 respectively, and the splitless injection mode was employed. Only a single ramp from 100 to 122 300 °C (held for 3.5 min) at 40 °C/min was set in the oven temperature program to perform However, there were no detectable signals for any of the nanoparticles. Under the tested 189 conditions, target analyte could not be adsorbed onto the selected nanoparticle or eluted from the surface of the nanoparticles. Therefore, Fig. 1 192 Results corresponding to the DLLME, SHS-LLME, and SFDF-LPME methods are given in 193 Fig. 1 .

Based on the peak values, the SFDF-LPME method was concluded to be the best 195 microextraction method among the three methods. All these methods yielded different results 196 based on ANOVA tests. SFDF-LPME was also superior to the other methods in terms of its 197 simplicity, rapidness, cost-effectiveness, and extraction efficiency. The spraying system used 198 in the SFDF-LPME method was developed in our previous study and detailed in the 199 corresponding report [26] . 

Sodium hydroxide was also used to remove the sulphate ions from the analyte sample. When 228 no sodium hydroxide was added, no analytical signal corresponding to the analyte was 229 detected. After this, 20, 40, and 80 g/kg of sodium hydroxide solutions were tested to 230 determine the optimum concentration. The optimized concentration was limited to 80 g/kg 231 because higher concentrations led to the formation of white precipitates between the aqueous 232 and organic layers. Fig. S1 shows that the highest peak areas were obtained for 80 g/kg of 233 sodium hydroxide.

To evaluate the effect of sodium hydroxide on analyte mass transfer from the aqueous applicability and accuracy. Human serum, urine, and saliva samples were first prepared. Upon 286 blank analyses, there was no detectable concentration of the analyte in any of the samples. As Table 1 . Analytical performance of all the systems and comparison with other studies.

Linear range LOD LOQ Correlation coefficient (R 2 ) Refs. LOD: limit of detection, LOQ: limit of quantification, GC-MS: gas chromatography-mass 468 spectrometry, UA-SFDF-LPME-GC-MS: ultrasonication-assisted spraying based fine droplet 469 formation-liquid phase microextraction-gas chromatography-mass spectrometry, DMSPE- 

Uncertainties (±) are represented by standard deviation (n=3)