Determination of carbapenem antibiotics using a purpose-made capillary electrophoresis instrument with contactless conductivity detection

Journal Pre-proof Determination of carbapenem antibiotics using a purpose-made capillary electrophoresis instrument with contactless conductivity detection Thi Ngoc Mai Pham, Thai Binh Le, Duc Dung Le, Tran Hung Ha, Ngoc Son Nguyen, Tien Duc Pham, Peter C. Hauser, Thi Anh Huong Nguyen, Thanh Duc Mai

PII:

S0731-7085(19)31673-5

DOI:

https://doi.org/10.1016/j.jpba.2019.112906

Reference:

PBA 112906

To appear in:

Journal of Pharmaceutical and Biomedical Analysis

Received Date:

12 July 2019

Revised Date:

20 September 2019

Accepted Date:

1 October 2019

Please cite this article as: Pham TNM, Le TB, Le DD, Ha TH, Nguyen NS, Pham TD, Hauser PC, Nguyen TAH, Mai TD, Determination of carbapenem antibiotics using a purpose-made capillary electrophoresis instrument with contactless conductivity detection, Journal of Pharmaceutical and Biomedical Analysis (2019), doi: https://doi.org/10.1016/j.jpba.2019.112906

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-1Determination of carbapenem antibiotics using a purpose-made capillary electrophoresis instrument with contactless conductivity detection

Thi Ngoc Mai Pham1, Thai Binh Le1, Duc Dung Le1, Tran Hung Ha2, Ngoc Son Nguyen1, Tien Duc Pham1, Peter C. Hauser3, Thi Anh Huong Nguyen1* and Thanh Duc Mai4*

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Department of Analytical Chemistry, Faculty of Chemistry, VNU University of Science, Vietnam

National University-Hanoi - 19 Le Thanh Tong, Hanoi, Vietnam Poison Control Center, Bach Mai Hospital, 78 Giai Phong road, Dong Da, Hanoi, Viet Nam

3

University of Basel, Department of Chemistry, Klingelbergstrasse 80, 4056 Basel, Switzerland

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2

Institut Galien Paris Sud, UMR 8612, Protein and Nanotechnology in Analytical Science (PNAS),

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CNRS, Univ. Paris-Sud, Univ. Paris-Saclay, 5 rue Jean Baptiste Clément, 92290 Châtenay-

[email protected];

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e-mail:

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Malabry, France

Highlights:

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[email protected]

Simple and affordable tool based on purpose-made CE-C4D for drug quality control.



CE-C4D methods for analyses of last-resort carbapenem antibiotics.

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 

Therapeutic monitoring of antibiotics in plasma samples from patients under intensive care.

-2Abstract In this study, the employment of a purpose-made capillary electrophoresis (CE) instrument with capacitively coupled contactless conductivity detection (C4D) as a simple and cost-effective approach for simultaneous determination of different carbapenem antibiotics is reported. The developed CE-C4D approach was for the first time applied for quality control of various pharmaceutical formulations in Vietnam, as well as for therapeutic monitoring of these antibiotics in plasma samples from patients under intensive care. Four of the most popular carbapenems in Vietnam, doripenem, meropenem, imipenem and ertapenem, were determined using an electrolyte

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composed of 10 mM Tris adjusted to pH 8.0 with acetic acid. The best detection limits achieved using the developed CE-C4D method were 0.36 mg/L and 0.45 mg/L for pharmaceutical and plasma samples, respectively. Good agreement between results from CE-C4D and the confirmation method

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(HPLC-PDA) was achieved, with a coefficient of determination (r2) for the two pairs of data of

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0.9967.

Keywords: carbapenems, antibiotics in plasma, capacitively coupled contactless conductivity

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detection (C4D), capillary electrophoresis (CE), purpose-made instrumentation.

-31. Introduction The carbapenems, structurally related to penicillins (penams), belong to the beta (β)-lactam antibiotics family, having an exceptionally broad spectrum of anti-microbiological activity. They also tend to be tolerant to the enzyme β-lactamase, on which the main mechanism of resistance of many bacteria is based. The analogs of carbapenem, including imipenem (1987), panipenem (1993), meropenem (1996), ertapenem (2001), biapenem (2002) and doripenem (2005) were indeed introduced to therapeutic use as the most potent antibiotics to kill harmful microorganisms which are resistant to penicillin and cephalosporin antibiotics. In particular, the carbapenems have been

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used as the last-resort antibiotics for patients in wards for infectious diseases and intensive care units who are seriously infected and / or multidrug-resistant. Unfortunately, the development of carbapenems-resistant microorganisms has been reported and is becoming an emerging threat [1],

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leading to a lack of therapeutic options to treat certain infections in humans and placing a high

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burden on healthcare systems and society [2]. , Such problem can be accelerated and worsened by the abuse of antibiotics as well as the unwanted misuse of low-quality or counterfeit antimicrobial

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products. . This raises a big concern in developing countries in general, and in Vietnam in particular. In these countries, the use of counterfeit antibiotics (the most counterfeited medicines

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worldwide [3]) as well as low-quality antibiotics is widespread. People can easily buy antibiotics for domestic use from retail pharmacies without medical prescription [4]. At the same time, the misuse of antibiotics by physicians, unskilled practitioners, and the public is common. Indeed, Vietnam, together with other developing countries in South East Asia, is regarded as a global

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hotspot for the emergence and spread of antimicrobial resistance [5]. Since the development of new antibiotics capable of fighting against microorganisms requires time and is expensive, measure to reduce the development of resistance to antibiotics, notably quality control of antimicrobial drugs and therapeutic monitoring of patients under antibiotic treatment, are of extreme importance. In another context, different studies have found that carbapenems are associated with a dose-related increase in the risk for seizure events [6, 7]. As this is highly linked with inadequate dose

-4adjustment in relation to kidney function, particular attention has to be paid during treatment therapies using carbapenems for inpatients under intensive care, especially those with renal failure symptoms. Reviews of adverse events observed over thousands of patients treated with carbapenems were already reported elsewhere [8, 9]. These situations lead to a need for simple and cost-effective analytical approaches for the determination of antibiotics in general, and carbapenems in particular, which are applicable in developing countries for drug quality control and therapeutic monitoring purposes. So far the analogues of carbapenems in biological and pharmaceutical matrices have mostly been determined with chromatographic methods, notably

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high-pressure liquid chromatography (HPLC) [10, 11]. The instrumental setups for HPLC however are not affordable for many hospital and clinical centers in developing countries with modest infrastructure and limited funding. On the other hand, capillary electrophoresis (CE) is an

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alternative for the development of simpler and less expensive analytical techniques. Indeed, the

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employment of CE has been reported for the determination of antibiotics in general and carbapenem in particular, with commercial CE instruments coupled with UV detection being predominantly

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used [12-16].

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Capacitively coupled contactless conductivity detection (C4D) is an alternative universal detection method for CE with different attractive features, notably high versatility, ease of construction and operation, low power consumption and the possibility of miniaturization. These features also allow its use in compact and / or purpose-made CE instruments which can also be built in low-cost

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versions [17]. An overview of the 20-year development of CE-C4D, covering details on working principles and applications, can be found in a recent review by Hauser and Kubáň [18] and the references listed therein. Indeed, the marriage between CE and C4D has been considered an economic and efficient approach for quality control of antibiotic formulations [19]. Nevertheless, the applications of CE-C4D for this purpose are still in their infancy and are outnumbered by those with CE-UV systems.

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This report is a continuation of our effort to introduce the use of low-cost purpose-made CE-C4D instruments in Vietnam. To the best knowledge of the authors, this has been the first time CE-C4D is applied for the determination of carbapenems. Herein, its applicability was demonstrated for quality control of 15 antibiotic drug formulations available in Vietnam and therapeutic monitoring of carbapenems concentrations in plasma samples from patients in an intensive care unit. The reliability of the CE-C4D approach developed for the analysis of a series of carbapenem antibiotics popularly used in Vietnam was validated through a cross-check using a standard HPLC-PDA

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method.

2. Experimental

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2.1. Chemicals and Materials

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All chemicals were of analytical or reagent grade and purchased from Merck (Germany), SigmaAldrich (USA) or TRC (Canada). Doripenem, meropenem sodium salt 98%, imipenem

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monohydrate 98%, ertapenem disodium 90 % were purchased from TRC. Individual stock solutions (1000 mg/L) of carbapenem-antibiotics were used for the preparation of standards. Chemicals used

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for preparation of CE-C4D buffers include: L-Arginine (Arg), Tris(hydroxymethyl)aminomethane (Tris), ascorbic acid (Asc), acetic acid (Ace) and lactic acid (Lac). Background electrolyte (BGE) solutions were prepared by addition of the selected acid (Lac, Ace or Asc) into a basic solution

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containing either Arg or Tris at desired concentrations.

Fused silica capillaries of 50 µm ID and 375 µm OD were obtained from BGB Analytik AG (Böckten, Switzerland). Before use, the fused silica capillaries were pre-conditioned with 1 M NaOH for 10 min and deionized water for 10 min prior to flushing with the buffer. The capillaries were then used continuously for successive analyses. Deionized water purified using a system from Water Pro (Labconco, Kansas City, MO, USA) was used for the preparation of all solutions and for

-6sample dilution if required. pH values of solutions were controlled with an HI 2215 Hanna Instruments pH meter (Woonsocket, RI, USA).

2.2. Instrumentation Detection was carried out with a commercial C4D (ER815, eDAQ, Denistone East, NSW, Australia). The compact CE instrument used was built in-house. High voltages of maximum 25 kV were provided with a miniature Spellman unit (UM25*4 - 12V, Pulborough, UK). The system was equipped with a microswitch to interrupt the high voltage (HV) when the lid was opened. The

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system was operated with 220-VAC power supply. More details on CE instrumentation can be found in our previous publications [20].

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2.3. Sampling and sample treatment

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15 antimicrobial drugs were purchased from central pharmacies in Hanoi, Vietnam. All tested antibiotics were injection powders, including Meronem 1 (500 mg) and 2 (1000 mg) (ACS Dobfar

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S.p.A., Italia), Meropenem Kabi 1 (500 mg) and 2 (1000 mg) (Fresenius Kabi Bidiphar, Germany), Merugold (Facta Farmaceutical SPA, Italia), Tiepanem (Facta Farmaceutical SPA, Italia), Pizulen

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(Demo S.A. Pharmaceutical Industry, Greek), Medozopen (Medochemie., Ltd., Cyprus), Nimidine (Anfarm Hellas S.A, Greek), Tienam (Merck Sharp & Dohme Corp., Germany), Lastinem (Venus Remedies., Ltd., India), Cepemid 1 (500 mg) and 2 (750 mg) (MD Pharco, Vietnam), Mixipem (Facta Farmaceutici S.p.A, Italia), Ivanz (Laboratoires Merck Sharp & Dohme-Chibret, France).

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These samples were treated as follow. The mass of each bottle was weighed precisely. The powder in the bottle was then collected and mixed well. The empty bottle was weighed again to determine the mass of powder in each bottle. About 1 % of the mixed powder was weighed and then transferred to a 10 mL volumetric flask which was then filled up to the mark with deionized water. The obtained solution was filtered through a 0.45 µm filter membrane and diluted when needed prior to injection to the CE-C4D instrument.

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The plasma samples from 5 different patients under intensive care using meropenem or imipenem were provided by the Poison Control Center (Bach Mai hospital). Antibiotic transfusion was implemented every 8 hours, with duration of 3 hours per transfusion. Meropenem was prepared in distilled water for injection, and then transferred into NaCl solution prior to transfusion. For monitoring meropenem or imipenem concentrations in plasma samples with CE-C4D, blood sampling (3 mL each time) was carried out before and at different times after antibiotic transfusion. After centrifugation at 4000 rpm for 10 min, 1 mL of plasma sample was taken for solid phase

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extraction (SPE) sample treatment whose protocol was adapted from [21]. The C18 columns

(Discovery® DSC-18, Supelco), pre-activated with 1 mL methanol and 3 mL deionized water (DI), were used for loading of plasma samples. Column washing after SPE was implemented with 1 mL

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DI, followed by elution of extracted meropenem with 2 mL methanol. For sample enrichment, the

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eluents were evaporated with nitrogen and then re-diluted into smaller volumes of methanol (50 µL to 500 µL, depending on initial carbapenem concentrations) prior to injection into the CE-C4D

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2.4. Analytical methods

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instrument.

The CE-C4D separation and quantification of four carbapenem-antibiotics, including doripenem, meropenem, imipenem and ertapenem were carried out using uncoated fused silica capillaries (ID of 50 μm and OD of 375 µm) with the total length of 60 cm and the effective length of 50 cm,

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respectively. Injection was carried out with the syphoning effect at a height of 20 cm for 20 seconds. The optimized BGE was composed of 10 mM Tris adjusted to pH 8.0 with Ace. Separations were carried out under a separation voltage of +20 kV. The cross-check operations were carried out using a HPLC-PDA protocol developed by the National Institute of Drug Quality Control (Vietnam), which was adapted from the United States Pharmacopeia (USP 40, 2017).

-83. Results and Discussion 3.1. Consideration on instrumental aspect Compared to commercial and costly instruments that may not be accessible to modestly-equipped laboratories, in-house-built CE-C4D indeed offers many advantageous features in terms of construction of inexpensive and transportable versions [22, 23], and flexible configuration setups [19]. After a 10-year testing of different CE-C4D prototypes in Vietnam, notably compact manual [24], semi-automated [25], fully automated single-channeled [26] and multiple-channeled [17] setups, a clear view on the user expectation could be established basing on their feedbacks on the

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drawbacks and positive features of different designs. In a laboratory where budget limitation plays an important role in decision making and skillful operators are not often available, the compact and manual systems were found to be the most adapted option thanks to the lowest cost and operational

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simplicity. Regarding CE instrumentation, Kuban et al. have presented a review detailing all steps

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required to construct a purpose-made CE system [27]. Note that a mechanical and electronic workshop, even modestly equipped, is often required for system construction and / or assembly. In

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the particular case for Vietnam, a good compromise between construction cost and performance can be made thanks to i) the introduction of Spellman miniature battery-operable high-voltage

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generation modules (see [25]) to replace the costly bi-polar ones employed in all commercial CE systems and ii) employment of pneumatic and / or mechanical components to replace electronic computer-controllable ones. While purpose-made / in-house built CE-C4D instrumentation is a good way to gradually bring this low-cost and simple approach to the population, continued efforts are

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still needed, especially for elimination of workshop facility requirement, to provide a drastic improvement of CE-C4D popularity outside the academic contexts.

3.2. CE-C4D methodology development for carbapenem antibiotics determination According to opinions of medical doctors from central hospitals, meropenem and imipenem are the carbapenems most frequently used in Vietnam. Depending on cases ertapenem and doripenem

-9could also be used from time to time in the treatment therapies. They can also be found in central pharmacies, nevertheless not as popular as meropenem and imipenem. Our CE-C4D method was therefore developed to cover all carbapenems that can be found in the pharmacies or in treatment therapies in Vietnam, regardless of the frequency of utilization or temporal availability. The structures and some characteristics of doripenem, meropenem, imipenem and ertapenem are detailed in Table 1. These compounds possess relatively high molecular weights and negative charges under basic conditions. Using an uncoated fused silica capillary, these large molecules with relatively low electrophoretic mobilities are pulled by the elevated electro-osmotic flow (EOF)

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towards the detector. Accordingly, optimization of working BGEs was implemented under basic conditions in order to provide sufficiently high EOF and at the same time low conductivity

generation for sensitive C4D signals. Different BGE compositions, using 10 mM Tris or Arg

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adjusted to pH 8.0 with either Asc, Ace or Lac were tested for electrophoretic separations of target

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carbapenem antibiotics (see Fig. 1). As can be seen, Tris/Ace offered the best concurrent separation of doripenem, meropenem, imipenem and ertapenem. Other combinations of Tris/Lac, Arg/Ace,

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Arg/Lac and Arg/Asc led to smaller peak heights (due to only small conductivity differences between the components of the BGEs and target analytes) and / or longer separation time. Further

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optimization was therefore carried out with BGEs composed of Tris/Ace. The pH of BGEs composed of a fixed concentration of Tris (10 mM) was then varied from 7.0 to 9.0 by adjusting the Ace amount for separation of target antibiotics (Fig. 2). This pH adjustment leads to a change of EOF magnitude, which in turns influenced the EOF-driven separation of the antibiotics. The best

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compromise between baseline separation and short analysis time was achieved at pH 8.0. Note that inorganic ions may be present in the samples injected to CE-C4D when deionized water is not available for sample dilution in resource-constrained facilities. With the tested BGEs, the peaks of carbapenems arrived right after the EOF whereas the abundant inorganic cations arrived before the EOF; and inorganic anions did not appear in the electropherograms during the separation time due to much retardation by EOF. The unwanted presence, sometimes inevitable, of interfering inorganic

-10cations and anions therefore would not pose a problem of peak overlapping to the target antibiotics in our case.

It was demonstrated in our previous work that peak heights in CE-C4D may also exhibit a dependence on the buffer concentration [28]. The Tris concentrations in the Tris/Ace BGEs (at pH 8.0) were therefore varied from 5 to 50 mM for signal height optimization (see Fig. 3). Satisfying signal-to-noise ratios and peak resolutions were obtained at Tris concentrations between 10 - 30 mM. Note that the target peaks, which were swept by the EOF, were retarded upon increase in Tris

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concentration which leads a reduction of the EOF. Note also that the migration times of target

analytes changed significantly during optimization of different parameters, i.e. BGE compositions (Fig. 1), pH of the BGEs (Fig. 2) and ionic strengths of the BGEs (Fig. 3). Indeed, when either of

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these parameters was modified, the EOF changed, leading to the variation of the migration times of

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target species whose apparent mobilities are influenced / driven by the EOF. The optimized BGE for determination of the carbapenem antibiotics within the shortest separation time is thus

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composed of Tris 10 mM adjusted to pH 8.0 with Ace. The salient performance data using the optimized CE conditions are shown in Table 2. The best detection limits achieved for the conditions

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employed were 0.36 ppm. Calibration curves were acquired up to 250 ppm with excellent linearity (R2 better than 0.998). The reproducibilities for peak areas and migration times were found to be better than 2.0 % and 1.7 % respectively.

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For analysis of carbapenem antibiotics in plasma samples, preconcentration by SPE was required to remove the plasma matrix and at the same time enrich the target analyte prior to injection into CEC4D. Different optimizations, including adjustment of the sample pH, volume of the washing solution, as well as eluent composition and volume were carried out with blank plasma spiked with 100 ppm meropenem (see Table 3). The best recovery of meropenem (97 %) after the sample treatment process was achieved with the pH of the plasma sample adjusted to 7.0, washing after

-11sample loading with 1 mL H2O and 2-time elution with 1 mL methanol. Further evaluations on recoveries for meropenem and imipenem were then carried out with these optimized conditions, using different tested concentrations (10, 20 and 30 ppm). Respective recoveries of 90 %, 105% and 114 % were achieved for meropenem, whereas those of 116%, 93% and 94 % were reached for imipenem, proving satisfied performance of the sample treatment protocol for both meropenem and imipenem.

3.3. Applications

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3.3.1. Quality control of carbapenem antibiotics in pharmaceutical formulations Carbapenems are the last-resort antibiotics, which render their costs relatively high and their use less popular by the population than the widely-used beta-lactam antimicrobial drugs. Carbapenems

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are therefore more often found in central pharmacies rather than in rural areas. Typical

electropherograms for the analyses of carbapenem antibiotics purchased from central pharmacies in

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March 2019 are shown in Fig. 4. As can be seen, a single carbapenem was present in the tested

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samples. Some kinds of counterfeiting for antibiotics, including a lack of active ingredients and wrong ingredients could therefore be excluded in these cases. In order to unambiguously identify the peaks, the standard addition was carried out. The CE-C4D approach was employed for further

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control of quantity of active ingredients in 15 carbapenem drugs. Cross-check with a confirmation method (HPLC-PDA) was carried out for some tested samples to confirm the reliability of the data obtained with CE-C4D. The coefficient of determination, r2, reaching 0.9967 and the deviation of

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less than 10.9 % observed in all samples for the two pairs of data (i.e. CE-C4D vs. HPLC-PDA) proved a very good agreement between the results obtained from two methods. Comparison of results revealed with CE-C4D and those registered on labels is shown in Table 4. Matching of antibiotics concentrations, with the difference between the results obtained with CE-C4D and those declared on the labels less than ± 5 %, was found for most of tested samples, except for Meronem and Ivanz where the difference exceeded the acceptable threshold of 10 %. Antimicrobial drugs containing doripenem were not found during the sample campaign; therefore no data regarding this

-12carbapenem were reported. Note also that for cross check operation with HPLC, samples had to be sent to the National Institute of Drug Quality Control (NIDQC). Some of the samples (Pizulen, Nimidine and Cepemid) were lost during delivery or administration at the NIDQC. The cross check results for these samples were therefore not obtained for table 4. This is indeed a fact / risk that may happen if all pharmaceutical samples have to be sent to central laboratories for quality control. Our proposed approach with CE-C4D that can be carried out directly in a local laboratory in the vicinity of sampling site can therefore help minimize this risk.

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3.3.2. Determination of carbapenems in plasma samples from patients under intensive care

The incorrect or indiscriminate use of carbapenems, as well as a poor quality of these antimicrobial drugs could facilitate the emergence of resistant pathogens, interfering therapy. The dosage of

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carbapenem antibiotics should be sufficient to inhibit the growth of micro-organism responsible for

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the infection in order to prevent the return of the disease, especially in the intensive care context. Depending on the health status of the inpatients (with particular attention paid to those having renal

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failure problems), carbapenem transfusion was implemented from 1 to 3 times per day, each time with a bottle containing 500 - 1000 mg antibiotics. Medical doctors will base on the plasma

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antibiotic levels to adjust the subsequent dosage for each patient. In our study, plasma samples from 5 inpatients who underwent carbapenem transfusion every 8 hours, two patients with imipenem treatment (500 mg per transfusion) and three others with meropenem treatment (1000 mg per transfusion) were analyzed with CE-C4D to monitor the diminution of plasma antibiotic levels.

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Electropherograms of plasma samples collected from one patient at different time after imipenem transfusion are shown in Fig 5A. Note that the peak corresponding to imipenem drifted significantly at different analysis times. Indeed, with the purpose-made instrument, the option of capillary thermostat was excluded to reduce construction cost. Fluctuation of migration times would therefore be encountered due to variation of the ambient temperature. The standard addition method was thus always implemented to confirm the peak identification and quantification, overcoming the

-13issue of migration time drifting between different samples which were induced from matrix and / or temperature variations. As can be seen, the peaks of imipenem well decreased over time, with only a little trace of imipenem remained after 5 h, demonstrating clearance of this antibiotic from the patient plasma. Similar observations were also found for patients treated with meropenem, except for higher remaining concentrations (up to 15 ppm) of plasma meropenem found after 5 h (see Fig. 5B). Note that the initial concentrations right after transfusion were much higher (almost 80 ppm) for patients treated with meropenem than for the one treated with imipenem (18 ppm). Cross verification revealed that for patients with risk of renal failure, the peak of carbapenem

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concentration in plasma should be around 20 ppm, then gradually decreasing to less than 2 ppm before the next transfusion. For those in good health conditions, the highest concentrations may reach 100 ppm with a transfusion of 1 g each time. The variation may also depend on the

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transfusion duration and intervals. The results obtained with our CE-C4D approach were also in

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accordance with the ranges and tendency of plasma imipenem and meropenem concentration evolution reported elsewhere using the standard HPLC method [29, 30]. The diminution of plasma

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meropenem / imipenem concentrations followed well the expected trend according to discussion with medical doctors. Note that no further experiments to provide replicate data (which otherwise

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would increase significantly the number of analyses per sample) were needed, as the increase in peak areas was found linear to the spiked concentrations, showing no aberrant results / tendency with the standard addition method.

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4. Conclusions

A purpose-made portable CE instrument coupled with an affordable commercial C4D was successfully applied for the separation and detection of different carbapenem antibiotics in pharmaceutical samples, serving for quality control and counterfeit drug identification purpose in Vietnam. For the treatment efficiency monitoring purpose, the method developed was also successfully applied to the analyses of carbapenems in plasma samples from patients under

-14intensive care. This has been the first time CE-C4D instrumentation and methodology is proposed as a cost-effective and simple solution for carbapenem analysis in the contexts of drug quality control and therapeutic treatment monitoring. Testing with a larger cohort of patient samples is anticipated in order to validate the clinical significance of our approach. The CE-C4D approach can be seen as a ‘low-cost’ alternative to the standard method and holds a potential for facile deployment in local laboratories with modest infrastructure and expertise for determination of antibiotics analyses in general and carbapenems in particular. Cross-check with the reference method proved the reliability of the results obtained with CE-C4D. This development is one of our continued efforts to participate

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in the actions of pharmaceutical quality control and screening of counterfeit medicines in emerging countries. The extension of CE-C4D application spectrum to other antibiotics is envisaged.

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Acknowledgements

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This research is funded by Vietnam National Foundation for Science and Technology Development

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(NAFOSTED) under grant number 104.04-2018.305.

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capillary electrophoresis analysis of antibiotics with capacitively coupled contactless conductivity detection, J. Pharm. Biomed. Anal. 158 (2018) 405-415.

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[20] A.P. Vu, T.N. Nguyen, T.T. Do, T.H. Doan, T.H. Ha, T.T. Ta, H.L. Nguyen, P.C. Hauser, T.A.H. Nguyen, T.D. Mai, Clinical screening of paraquat in plasma samples using capillary electrophoresis with contactless conductivity detection: Towards rapid diagnosis and therapeutic treatment of acute paraquat poisoning in Vietnam, J. Chromatogr. B 1060 (2017)

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[21] D.G. Musson, K.L. Birk, A.M. Cairns, A.K. Majumdar, J.D. Rogers, High-performance liquid chromatographic methods for the determination of a new carbapenem antibiotic, L-749,345, in human plasma and urine, J. Chromatogr. B 720(1) (1998) 99-106. [22] A.V. Schepdael, Recent Advances in Portable Analytical Electromigration Devices, Chromatography 3(2) (2016) doi:10.3390/chromatography3010002.

-17[23] A.P. Lewis, A. Cranny, N.R. Harris, N.G. Green, J.A. Wharton, R.J.K. Wood, K.R. Stokes, Review on the development of truly portable and in-situ capillary electrophoresis systems, Meas. Sci. Technol. 24(4) (2013). [24] T.A.H. Nguyen, V.R. Nguyen, D.D. Le, T.T.B. Nguyen, V.H. Cao, T.K.D. Nguyen, J. Saiz, P.C. Hauser, T.D. Mai, Simultaneous determination of rare earth elements in ore and anticorrosion coating samples using a portable capillary electrophoresis instrument with contactless conductivity detection, J. Chromatogr. A 1457 (2016) 151-158. [25] T.A.H. Nguyen, T.N.M. Pham, T.T. Doan, T.T. Ta, J. Sáiz, T.Q.H. Nguyen, P.C. Hauser, T.D.

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Mai, Simple semi-automated portable capillary electrophoresis instrument with contactless conductivity detection for the determination of beta-agonists in pharmaceutical and pig-feed samples, J. Chromatogr. A 1360(0) (2014) 305-311.

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[26] T.D. Mai, T.T.T. Pham, J. Sáiz, P.C. Hauser, Portable Capillary Electrophoresis Instrument

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with Automated Injector and Contactless Conductivity Detection, Anal. Chem. 85(4) (2013) 2333-2339.

(2019) 65-78.

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[27] P. Kubáň, F. Foret, G. Erny, Open source capillary electrophoresis, Electrophoresis 40(1)

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[28] T.D. Mai, P.C. Hauser, Study on the interrelated effects of capillary diameter, background electrolyte concentration, and flow rate in pressure assisted capillary electrophoresis with contactless conductivity detection, Electrophoresis 34(12) (2013) 1796-1803. [29] A. Wen, Z. Li, J. Yu, R. Li, S. Cheng, M. Duan, J. Bai, Clinical Validation of Therapeutic

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Drug Monitoring of Imipenem in Spent Effluent in Critically Ill Patients Receiving Continuous Renal Replacement Therapy: A Pilot Study, PLOS ONE 11(4) (2016) e0153927.

[30] P. Hanberg, K. Oebrink-Hansen, A. Thorsted, M. Bue, M. Tottrup, L.E. Friberg, T.F. Hardlei, K. Soballe, J. Gjedsted, Population Pharmacokinetics of Meropenem in Plasma and Subcutis from Patients on Extracorporeal Membrane Oxygenation Treatment, Antimicrob. Agents Chemother. 62(5) (2018) e02390-17.

-18Figure captions:

Fig. 1.

Electropherograms for the separation of carbapenems (50 ppm) with different BGE compositions. Other CE-C4D conditions: voltage: +20 kV; capillary: uncoated fusedsilica, 50 µm id, Lt = 60 cm (Leff = 50 cm).

Fig. 2.

Electropherograms for the separation of carbapenems with BGEs composed of Tris 10

Fig. 3.

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mM adjusted to pH from 7.0 to 9.0 with acetic acid. Other conditions as for Fig. 1.

Electropherograms for the separation of carbapenems with BGEs composed of Tris 5 - 50

Fig. 4.

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mM adjusted to pH 8.0 with acetic acid. Other conditions as for Fig. 1.

Electropherograms for separation of carbapenems in different pharmaceutical

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formulations with CE-C4D. CE conditions: BGE Tris 10 mM adjusted to pH 8.0 with

Fig. 5.

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acetic acid. Other conditions as for Fig. 1.

A) CE-C4D electropherograms for determination of imipenem in plasma samples collected from an inpatient at different times after antibiotic transfusion. CE conditions:

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BGE Tris 10 mM adjusted to pH 8.0 with acetic acid. Other conditions as for Fig. 1. B) Variation of carbapenem levels in plasma samples from in-patients under intensive care at different times after transfusion.

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Fig 1

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Fig 2

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Fig 3

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Fig 4

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Fig 5A

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Fig 5B

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Table 1: Structures and characteristics of the target carbapenem antibiotics Structure

Characteristics C15H24N4O6S2- M = 420,504 g/mol

Doripenem

Crystalline powder, with colour from white to somewhat yellowish. Soluble in water, slightly soluble in methanol, and virtually insoluble in ethanol. pKa=3.54; pKa = 9.51 Usually used for intravenous injection. C17H25N3O5S - M= 383,464 g/mol

pKa=3.47; pKa = 9.39

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C22H25N3O7S - M = 475,516 g/mol

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Meropenem

Crystalline powder, with colour from white to somewhat yellowish; slightly soluble in water, virtually insoluble in ethanol; insoluble in ether and acetone

Ertapenem

White crystalline powder, hygroscopic; soluble in water, insoluble in ethanol, isopropyl acetate and tetrahydrofuran.

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pKa=3.22; pKa = 9.03

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Usually used for intravenous injection.

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White crystalline powder, hygroscopic; Solubility of imipenem: 1g/1000 mL H2O or 1g/2000 mL methanol, insoluble in ethanol, dimethylformamide and dimethylsulfoxide pKa=3.63; pKa = 10.88

Usually used for intravenous injection.

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Imipenem

C12H17N3O4S - M = 299,347 g/mol

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Table 2. Salient performance data for the determination of carbapenem antibiotics with the purpose-made compact CE-C4D system. Conditions: BGE solution: 10 mM Tris / Ace (pH 8.0); voltage: +20 kV; capillary: uncoated fused-silica, 50 µm id, Lt = 60 cm (Leff = 53 cm). LOD a

LOQ

Calibration range

Linearity

(mg/L)

(mg/L)

(mg/L) b

(R2)

Peak area

Migration time

Doripenem

1.2

3.5

3.5 ÷ 150

0.9994

1.5

0.8

Meropenem

0.7

2.4

3.0 ÷ 250

0.9994

1.9

1.2

Ertapenem

1.9

6.3

7.0 ÷ 150

0.9989

1.6

1.6

Imipenem

0.4

1.2

1.5 ÷ 100

0.9988

1.6

0.7

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b

Based on peak heights corresponding to 3 times the baseline noise

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a

RSD % (n = 6)

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Table 3. Recovery of meropenem spiked in blank plasma after the SPE sample treatment. Each SPE optimization was carried out with 1 mL plasma sample. The recovery was calculated according to the CE-C4D peaks of meropenem from the same spiked plasma sample before and after SPE sample treatment. CE conditions as in table 2.

ammoniac solution

Washing after SPE

Elution

Recovery (%)

3

1mL H2O

1mL MeOH : H2O 7:3

10.7

5

1mL H2O

1mL MeOH : H2O 7:3

27.8

6

1mL H2O

1mL MeOH : H2O 7:3

34.9

7

1mL H2O

1mL MeOH : H2O 7:3

79.0

8

1mL MeOH : H2O 7:3

78.6

9

1mL H2O 1mL H2O

1mL MeOH : H2O 7:3

70.0

7

1 x 1mL H2O

1mL MeOH : H2O 7:3

79.0

7

2 x 1mL H2O

1mL MeOH : H2O 7:3

67.9

7

3 x1 mL H2O

1mL MeOH : H2O 7:3

53.3

7

1mL H2O

7

1mL H2O

7

1mL H2O

7 7

1mL ACN

21.6

1mL MeOH : H2O 7:3

79.0

1mL MeOH

85.0

1mL H2O

2 x 1mL MeOH*

97.0

1mL H2O

1mL ACN

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* Elution was carried out twice, each time with 1 mL MeOH

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Table 4. Concentrations of carbapenems in pharmaceutical samples determined with CE-C4D and cross-check results with HPLC-PDA

Sample Name

No

Concentration (mg/bottle) Carbapenem CE-C4D

Label information

HPLC

CE-HPLC CE-Label Deviation Deviation (D1, %)* (D2, %)**

Meropenem

498 ± 3.1

491

500

1.4

-0.4

2 Meropenem Kabi 2

Meropenem

914 ± 7.3

934

1000

-2.1

-8.6

3 Merugold

Meropenem

975 ± 6.9

930

1000

4.8

-2.5

4 Tiepanem

Meropenem

980 ± 7.1

884

5 Meronem 1

Meropenem

489 ± 3.8

442

6 Meronem 2

Meropenem

887 ± 5.8

934

7 Pizulen

Meropenem

486 ± 2.9

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8 Medozopen

Meropenem

937 ± 8.5

9 Cepemid 1

Imipenem

469 ± 2.7

10 Cepemid 2

Imipenem

11 Nimidine

Imipenem

12 Tienam 13 Lastinem

-2.0

500

10.6

-2.2

1000

-5.1

-11.3

500

-

-2.8

951

1000

-1.5

-6.3

482

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500

-2.7

-6.2

735 ± 5.3

-

750

-

-2.0

495 ± 3.5

-

500

-

-1.0

Imipenem

515 ± 3.9

506

500

1.8

3.0

Imipenem

519 ± 4.5

508

500

2.2

3.8

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1 Meropenem Kabi 1

14 Mixipem

Imipenem

478 ± 3.5

481

500

-0.6

-4.4

15 Ivanz

Ertapenem

867 ± 4.8

883

1000

-1.8

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* D1 % is the deviation of the result obtained with CE-C4D from that with the standard reference method; D % = {(RCE-C4D - Rreference) / Rreference} × 100 % ** D2 % is the deviation of the result obtained with CE-C4D from that indicated on the label; D2 % = {(RCE-C4D - Rlabel) / Rlabel} × 100 %