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Simple and rapid method for analysis of urinary vancomycin using solid phase extraction and fluorescence spectroscopy

Simple and rapid method for analysis of urinary vancomycin using solid phase extraction and... Vancomycin ( VCM) is an antimicrobial that is recommended for therapeutic drug monitoring ( TDM) for maintaining the efficacy and safety of treatment. The trough monitoring has been used to guide VCM dosing regimens. However, newer guidelines recommend the use of area under the curve/minimum inhibitory concentration (AUC/MIC)-guided vancomycin dosing, and there is a need for easier and more frequent measurements of VCM concentrations. There- fore, in this study, we developed a simple and rapid analytical method for measuring urinary VCM by combining solid- phase extraction and fluorescence analysis. Urine samples are easier and less invasive than blood samples. In addition to the therapeutic range of blood VCM, this method was also able to measure 0.01–1 mg/mL, which is the concentra- tion range of urinary VCM. The accuracy of 10, 20, and 30 μg/mL VCM solutions were between 93.18 and 109.76%. The relative standard deviation (RSD) of intra-day and inter-day analysis were less than 6.25% and 6.28%, respectively. Since this method does not use large equipment, it is expected to be better suited for clinical use. Keywords Fluorescence analysis, Vancomycin, Urine, Dosing, Therapeutic drug monitoring concentration (AUC/MIC) (Tkachuk et  al. 2018; Revilla Introduction et  al. 2010); however, trough monitoring is still a pre- Vancomycin (VCM), with a broad antimicrobial spec- ferred suitable indicator for the adjustment of VCM trum, is used as a first-line drug against methicillin- dose because of easy measurement (Liu et al. 2011; Kul- resistant Staphylococcus aureus (MRSA) (Cafferkey et al. lar et  al. 2011). Subsequent studies have reported about 1982). However, overdose of VCM may cause nephro- trough monitoring not being an ideal substitute predic- toxicity and acoustic disturbance (Jeffres et  al. 2007). tor, and the latest guidelines recommend AUC/MIC for Therefore, VCM is recommended for therapeutic drug guiding VCM dosing regimens (Rybak et  al. 2020; Mohr monitoring (TDM), and its dosage is adjusted based on and Murray 2007). Accurate determination of AUC its blood concentration (Patel et al. 2011). requires collecting blood samples numerous times from Based on pharmacokinetic/pharmacodynamic analy- patients and measuring concentrations over time, which sis, the most suitable predictor for the efficacy and safety is burdensome on patients and healthcare profession- of VCM is area under the curve/minimum inhibitory als. Therefore, there is a strong demand for a conveni - ent non-invasive or less invasive technique for collecting samples and obtaining necessary information for guiding *Correspondence: Masaru Kato VCM dosing regimens. masaru-kato@umin.ac.jp When VCM is administered intravenously, almost all Devision of Bioanalytical Chemistry, Graduate School of Pharmacy, of it is rapidly excreted in urine as an unaltered drug Showa University, 1-5-8 Hatanodai, Shinagawa-Ku, Tokyo 142-8555, Japan Department of Pharmaceutical Sciences, School of Pharmacy, Showa (Moellering et  al. 1981). Therefore, many studies have University, 1-5-8 Hatanodai, Shinagawa-Ku, Tokyo 142-8555, Japan been reported measuring VCM concentrations in the © The Author(s) 2023. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http:// creat iveco mmons. org/ licen ses/ by/4. 0/. Oshima et al. AAPS Open (2023) 9:2 Page 2 of 8 urine instead of blood (Shokouhi et al. 2017; Baranow- Urine sample analysis ska et  al. 2009, 2010; Cass et  al. 2011; Javorska et  al. Urine was obtained from healthy five male volunteers 2017). Thus, VCM can be measured easily and fre - with ages from 20 to 50  s (median age, 41). Approxi- quently in urine instead of blood for guiding VCM dos- mately 50 mL voluntary urine was collected and centri- ing regimens. This will also aid in obtaining accurate fuged by centrifugate (MX-300, Tomy Seiko Co., Ltd., AUC values, which may be useful for designing more Tokyo, Japan) at 600 × g for 10 min to remove the cells precise dosing regimens. The advantages of using urine (Kato et  al. 2020). The supernatant of the urine was as a sample over blood are (1) self-collection by the stocked in a refrigerator. A concentration of 10 mg/mL patient; (2) collection in large quantities (1.5 L/day); (3) VCM was prepared in water. The VCM solution was urine is usually discarded and there is almost no addi- added to the stocked urine just before the measure- tional burden on the patient; (4) using a urinary cathe- ment. MonoSpin Ph (GL Sciences Inc., Tokyo, Japan) ter, it is possible to collect all urine excreted in a certain was preconditioned by centrifugate (7780II, KUBOTA period of time; and (5) there is less chance of contami- Corporation, Tokyo, Japan) at 10,000 × g for 1  min nation because the substance filtered by the glomerulus with methanol and water as per the manufacturer’s and not reabsorbed by the renal tubules is excreted. protocol. Then, 600 µL of urine sample was added to Various reports on the measurement of urinary VCM the preconditioned MonoSpin Ph and washed four have shown a good correlation between blood and urine times with 550 µL phosphate-buffered saline (PBS). concentrations (Shokouhi et  al. 2017), and urine may The captured VCM was eluted in 50 µL eluent (citrate serve as an alternative sample for guiding VCM dos- buffer (pH 2): methanol; 90:10). To the eluted solution, ing regimens. Additionally, VCM concentration in 24-h 270 µL DMSO was added, followed by 300 µL of the urine samples has been reported to be much higher than mixture solution was moved to a 96-well plate for fluo- the blood VCM concentration (several hundred µg/mL rescence analysis. to 1  mg/mL) (Javorska et  al. 2017). For measuring the concentration of VCM in blood, immunoassay is fre- HPLC measurement quently used clinically (Sattur et al. 2000; Vila et al. 2007), HPLC analyses were performed using a Hitachi and recently, a method using high performance liquid LaChrom Ultra series (Hitachi High-Tech Science chromatography (HPLC) has also been developed which Corporation, Tokyo, Japan) consisting of two L-2160 is used for short-time analysis in clinical use (Farin et al. U LaChrom Ultra pumps, an L-2200U LaChrom auto 1998; Trujillo et al. 1999; Javorska et al. 2016). However, sampler, an L-2455U LaChrom diode array detector, these analytical methods require expensive equipment, and an HPLC system organizer. A Resolve of 5-µm and it is difficult to measure high concentrations of VCM Spherical C18 column (3.9 mm × 150 mm, Waters, (several 100  mg/mL or more) accurately using common Milford, MA, USA) was used. Mobile phase A had 20 competitive immunoassays. Therefore, in this study, we mM formic acid/ammonium formate and mobile phase developed a new method for measuring urinary VCM B had 100% methanol. The gradient elution program that can be easily used in a clinical setup and can meas- of the mobile phases was as follows: 5–10% (B) from ure a wide range of VCM concentrations. 0 to 5 min. Flow rate was 1 mL/min. The injection volume was 10 µL, and a fluorescence detector with an excitation wavelength (ex) at 290 nm and emission Methods and materials wavelength (em) of 330 nm was used for detection. All Chemicals and reagents samples were filtered using a Millex-LG syringe filter Ammonium formate, citric acid anhydrous, N,N-dimeth- (pore size, 0.2 µm, Millipore) before analysis. ylformamide (DMF), dimethyl sulfoxide (DMSO), etha- nol, formic acid, methanol, trisodium citrate dihydrate, Measurement of three‑dimensional (3D) fluorescence and VCM were purchased from Fujifilm Wako Pure spectrum Chemical Corporation, Ltd. (Osaka, Japan). Glycerol The 3D fluorescence spectra were measured using was purchased from Nacalai Tesque, Inc. (Kyoto, Japan). a fluorescence spectrometer (F7100, Hitachi High Sulbactam sodium/ampicillin sodium and piperacil- Technologies Corporation, Tokyo, Japan). Excitation lin/tazobactam were obtained from Meiji Seika Pharma spectra were recorded in the 200–500  nm range with Co., Ltd. (Tokyo, Japan). Meropenem and ceftriaxone emission measurements from 200 to 500  nm. The slit were obtained from NIPRO Corporation (Osaka, Japan). width was 5  nm. The 3D spectra were shown using Cefepime was obtained from Sandoz K.K. (Tokyo, Japan). SpectAlyze software package (Dynacom Co., Ltd., Chiba, Japan). Oshima  et al. AAPS Open (2023) 9:2 Page 3 of 8 Measurement of fluorescence spectrum and intensity VCM has fluorescence intensity in the ultraviolet region The fluorescence of VCM was measured using a mul - at an excitation of 280–300  nm and emission of 290– tiplate reader (SH-9000, Corona Electric Co., Iba- 340 nm. Next, when the 3D-fluorescence spectrum of the raki, Japan). Fluorescence spectrum was obtained in urine of a healthy volunteer was measured, there were the emission wavelength from 300 to 400  nm when two fluorescence peaks, the first with an excitation of excited at 290 nm. Fluorescence intensity was obtained 250–290  nm and emission of 325–400  nm, and the sec- in the emission wavelength of 340  nm when excited at ond with an excitation of 310–360  nm and emission of 290 nm. 360–400  nm (Supporting Fig.  1). For accurate measure- ments of VCM concentration in the urine for clinical use, Measurement of VCM in a clinical laboratory it is necessary to remove fluorescent substances (amino The clinical laboratory used Emit 2000 Vancomycin acids, flavins, NADH, and others) present in urine Assay kit (Siemens Healthineers, Erlangen, Germany) (Kušnír et al. 2005) because the first fluorescence peak in and BioMajesty JCA-BM8000 series (JEOL Ltd., Tokyo, the urine sample partially overlaps with the fluorescence Japan) for the analysis. The measurements were per - of VCM. formed as described in the manufacturer’s protocol. Hence, we first determined the purification conditions for VCM from urine using a solid phase extraction (SPE) Results and discussion cartridge. A phenyl group-modified SPE cartridge was Fluorescence spectroscopy is a very sensitive and selec- selected because VCM has five aromatic rings. VCM was tive technique. Therefore, it is an ideal method for the applied to the cartridge, and each fraction was eluted in detection of trace substances in biological fluids. In PBS solution and citrate buffer (pH 2) containing 10, 15, addition, simple and inexpensive fluorometers are now 20, and 60% methanol. The eluted samples were analyzed available in the market. Supporting Fig.  1 shows the 3D by HPLC. VCM was not detected in the flow-through fluorescence spectrum of VCM at the excitation (ex.) fractions, indicating that VCM was efficiently captured wavelength range of 200–500  nm and emission (em.) in the SPE cartridge. Also, in the eluted fractions, VCM wavelength range of 200–500 nm. Warm colors (Ex: red) was detected at 9 min only in the eluted fraction of 10% indicate a strong fluorescence intensity, whereas cold methanol solution, and not in the other fractions (Fig. 1). colors (Ex: blue) indicate a weak fluorescence intensity. Specifically, urinary VCM was purified by passing the Fig. 1 Chromatograms of fractionates and 100 μg/mL VCM solutions. Column: Resolve 5-µm spherical C18 column (3.9 mm × 150 mm), mobile phase A: 20-mM formic acid/ammonium formate, B: 100% methanol, gradient program: 5–10% (B) from 0 to 5 min, flow rate: 1 mL/min, detection: fluorometry (ex/em. 290/330 nm) Oshima et al. AAPS Open (2023) 9:2 Page 4 of 8 Fig. 2 3D fluorescence spectra of washed solution. Excitation: 240–380 nm, emission 260–400 nm; the red circle indicates region of VCM fluorescence signal urine sample through an SPE cartridge, washing the car- washings (Fig.  2). This signal was derived from solvent- tridge repeatedly with PBS, and performing final elution derived Raman scattered light. in a 10% methanol solution. Then, we examined solvents that reduced the influence Next, the number of washings with PBS required for of the Raman scattering of the solvent itself. Fluorescence purification of the urine sample was examined. A urine spectra of five solvents (DMF, DMSO, methanol, glycerin, sample was placed on a cartridge, washed repeatedly and ethanol) that were excited at 290  nm are shown in with PBS, and the 3D fluorescence spectrum of the eluted Supporting Fig.  2. The scattered light of DMSO was the fractions were measured (Fig. 2). In the first washed frac - smallest among them. tion, strong fluorescence was detected near excitation of In order to completely eliminate the scattered light of 290  nm and emission of 330  nm (red circle). This indi - the eluent, the eluent must be completely evaporated and cates the elution of endogenous substances that exhibit redissolved in DMSO before fluorescence analysis. How - similar fluorescence properties to those of VCM in ever, drying of 300 μL of 10% methanol aqueous solution this fraction. The fluorescence in the region decreased is unsuitable for clinical use due to the extra time and with repeated washing, and no significant change was equipment required. Furthermore, there is a possibil- observed even after 4 or more washes. However, fluores - ity that the decomposition of VCM, which is unstable cence signal around 300–340  nm was observed and the in the solution, may occur in the drying process (White intensity remained constant regardless of the number of et  al. 1988; Serri et  al. 2017; Cao et  al. 2018). Therefore, Oshima  et al. AAPS Open (2023) 9:2 Page 5 of 8 we thought that if the volume of the eluent from the was approximately 80%, the fluorescence intensity was 10 cartridge was reduced as much as possible, and DMSO times stronger than that of the sample without DMSO, was added without drying, it would be easier to use in and even if the DMSO proportion was increased further, the clinical setup. The volume of the cartridge of SPE is the increase in the fluorescence intensity was not very approximately 20.8  μL (2.1 × 2.1 × π × 1.5 mm ) and its high. Based on the above results, we determined the pro- porosity is 80%; therefore, the void volume of the car- cedure of the analysis of urinary VCM as follows: urine tridge is 16.6 µL. In other words, it was expected that sample was centrifuged for removing cells and debris. most of the VCM captured on the cartridge would be Then, 600 µL supernatant was applied to Ph-modified eluted using an eluent several times as large as 16.6 µL. SPE cartridge and washed four times with 550 µL PBS. Hence, we loaded the cartridge with the same amount The captured VCM was eluted in 50 µL of 10% methanol, (90 µg) of VCM and measured the fluorescence of VCM and then, 270 µL of DMSO was added. The fluorescence in each fraction that was eluted in different volumes intensity (excitation at 290  nm, emission at 340  nm) (10–300 µL) of the eluent (Fig.  3a). As a result, it was of 300 µL of the mixture solution was measured by a found that some VCM was eluted even in 10 µL of elu- fluorometer. ent, and the amount of eluted VCM increased as the vol- First, we made a calibration curve in the range of ume of eluent increased. Approximately 95% of the VCM 0–50  µg/mL, which is necessary for measuring the was eluted when 300 μL eluent was used and only 36.1% therapeutic concentration range of VCM in the blood VCM was eluted by 50 μL eluent. However, their variabil- (Fig. 4). The samples were used where VCM was added ity was 3.10 and 1.51%, respectively, indicating quantita- to PBS or urine from healthy volunteers. As a result, tive elution even with a small amount of eluent. the calibration curves for urine and PBS were similar, Then, the effect of the mixing ratio of 10% metha - suggesting that contaminants in urine did not interfere nol solution, which is the eluent, and DMSO on the with the measurement of VCM. The correlation coeffi - fluorescence intensity of VCM was investigated. The cient of the calibration curve of urine and PBS was 0.94 fluorescence spectra of VCM were measured when the and 0.94, respectively. The validation data (intra-day proportion of DMSO in the measurement solvent was and inter-day reproducibility at three different concen - changed in the range of 0–97% (Fig.  3b). Even with- trations (10, 20, and 30  μg/mL) are shown in Table  1. out the DMSO solution, the fluorescence intensity The accuracy of 10, 20, and 30 μg/mL VCM solutions increased slightly by the addition of VCM. The intensity were between 93.18 and 109.76%. The RSD of intra-day was increased by increasing the proportion of DMSO and inter-day analysis were less than 6.25% and 6.28%, and it reached the maximum when the DMSO propor- respectively. Therefore, it was concluded that this assay tion was 97%. However, when the proportion of DMSO was applicable for quantitative analysis. To examine Fig. 3 a The relationship between elution volume and quantity of VCM, b relationship between ratio of DMSO to 10% methanol solution and quantity of VCM. Fluorescence spectra were obtained when excited at 290 nm. The sample volume was adjusted to 300 μL by adding 10% methanol solution before the fluorescence analysis Oshima et al. AAPS Open (2023) 9:2 Page 6 of 8 during the measurement, because it was reported that VCM is unstable in aqueous solutions (White et  al. 1988; Serri et  al. 2017; Cao et  al. 2018). Supporting Fig.  4 shows chromatograms of VCM samples immedi- ately after preparation and samples left at 37 ºC for 1 h that was more severe than the measurement conditions of 20 ºC for 30  min. The peak intensity of VCM did not change and no new peak derived from degradants appeared due to the storage. Hence, the VCM degrada- tion was neglectable during the measurement. Furthermore, since it has been reported that more than 100  µg/mL of VCM exists in urine, a calibration curve was also made using urine samples adjusted to VCM concentrations of 100, 300, 500, and 1000  µg/ mL (Vila et  al. 2007). A good calibration curve (corre- lation coefficient 0.97) was obtained even in the high- concentration region (Supporting Fig.  5). To confirm Fig. 4 Calibration curve for VCM in PBS and urine solution with the reliability of the developed method, we compared VCM concentrations from 0 to 50 μg/mL. Square and cross indicates PBS and urine, respectively. Error bars represent CV of three the measured values of the same samples with that by measurements the method currently used in clinical practice. Figure 5 shows the measurements of six different urine sam - ples using the developed method and EMIT method the effect of co-administrated drugs for the analy - by a clinical laboratory (Chen et  al. 2020). All samples sis, fluorescence of 5 frequently administrated drugs showed similar results, indicating reliability of the (sulbactam sodium/ampicillin sodium, piperacillin/ developed method for the analysis of urinary VCM. tazobactam, meropenem, ceftriaxone, and cefepime) Although urine from healthy subjects were used in was examined (Raverdy et  al. 2013). Supporting Fig.  3 this study, in order to use this method in clinical prac- shows the 3D fluorescence spectra of the 200  mg/mL tice, it is necessary to verify its effectiveness using co-administrated drug solutions. Strong fluorescence patient urine. The composition of patient specimens signal was observed from sulbactam sodium/ampicil- may differ significantly from those of healthy individu - lin sodium, and weak signal was observed from mero- als. One advantage of the developed method is that it is penem. Other co-administered drugs did not show a non-destructive method. In other words, if the meas- fluorescence signals in the range of 200 to 600  nm ured value by the developed method is abnormal, it can when excited from 200 to 600 nm. Although sulbactam be re-measured by other existing methods to confirm sodium/ampicillin sodium and meropenem showed the accuracy of the value. Therefore, we will measure fluorescence signals, the wavelength of fluorescence patient specimens using this method and obtain the signals were different from that of VCM. Hence, these AUC/MIC necessary for VCM administration design in co-administered drugs did not interfere with the VCM our next study. measurement. The degradation of VCM was evaluated Table 1. Inter-day and intra-day accuracy and validation at three different concentrations Oshima  et al. AAPS Open (2023) 9:2 Page 7 of 8 Fig. 5 Comparison of measurement values by the developed method and a clinical laboratory using EMIT method. EMIT method used Emit 2000 Vancomycin Assay kit and BioMajesty JCA-BM8000 series Funding Conclusions This work was supported by the JSPS KAKENHI. In this study, we developed a simple and rapid method for the analysis of urinary VCM. Since the method uses a Availability of data and materials Not applicable. centrifuge and fluorometer as equipment, it is cost-effec - tive compared to the other existing methods and is suit- Declarations able for clinical use. The developed method was able to measure up to 1000  μg/mL VCM, which is the assumed Ethics approval and consent to participate amount in urine samples, in contrast to the one currently All clinical studies involving human urine samples were conducted in adher- ence to the procedure approved by the Human Ethics Committee of the used in clinical laboratories (100  μg/mL). In the future, Showa University (approval no. 315), and informed consent was received from the use of this method to measure VCM in urine samples the volunteers. could help us clarify the relationship between urinary Competing interests VCM concentration and AUC/MIC and also guide VCM The authors declare that they have no competing interests. dosing regimens. Acknowledgements Received: 8 October 2022 Accepted: 9 January 2023 We acknowledge Prof. T. Sasaki, Prof. Y. Niki, Prof. I. Tokimatsu, Dr. T. Takuma, Dr. K. Karasawa, Dr. S. Murayama, Dr. Y. Naito, Mrs. K. Nakane, Dr. Y. Odanaka, and Mr. S. Maruyama for their assistance in this study. Authors’ contributions References Y.O. contributed conceptualization, methodology, formal analysis, investiga- Baranowska I, Markowski P, Baranowski J (2009) Development and validation tion, resources, writing original draft, writing-review & editing, visualization, of an HPLC method for the simultaneous analysis of 23 selected drugs supervision, and project administration. M. H. & M. M. contributed methodol- belonging to different therapeutic groups in human urine samples. Anal ogy, formal analysis, investigation, resources, writing original draft, writing- Sci 25:1307–1313. https:// doi. org/ 10. 2116/ anals ci. 25. 1307 review & editing, and visualization. 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Br J Clin Pharmacol 70:201–212. https:// doi. org/ 10. 1111/j. 1365- 2125. 2010. 03679.x Rybak MJ et al (2020) Therapeutic monitoring of vancomycin for serious methicillin-resistant Staphylococcus aureus infections: a revised con- sensus guideline and review by the American Society of Health-System Pharmacists, the Infectious Diseases Society of America, the Pediatric Infectious Diseases Society, and the Society of Infectious Diseases http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png AAPS Open Springer Journals

Simple and rapid method for analysis of urinary vancomycin using solid phase extraction and fluorescence spectroscopy

Oshima, Yuki; Hori, Mizuki; Matsumoto, Miyu; Kato, Masaru
AAPS Open , Volume 9 (1) – Feb 6, 2023
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Abstract

Vancomycin ( VCM) is an antimicrobial that is recommended for therapeutic drug monitoring ( TDM) for maintaining the efficacy and safety of treatment. The trough monitoring has been used to guide VCM dosing regimens. However, newer guidelines recommend the use of area under the curve/minimum inhibitory concentration (AUC/MIC)-guided vancomycin dosing, and there is a need for easier and more frequent measurements of VCM concentrations. There- fore, in this study, we developed a simple and rapid analytical method for measuring urinary VCM by combining solid- phase extraction and fluorescence analysis. Urine samples are easier and less invasive than blood samples. In addition to the therapeutic range of blood VCM, this method was also able to measure 0.01–1 mg/mL, which is the concentra- tion range of urinary VCM. The accuracy of 10, 20, and 30 μg/mL VCM solutions were between 93.18 and 109.76%. The relative standard deviation (RSD) of intra-day and inter-day analysis were less than 6.25% and 6.28%, respectively. Since this method does not use large equipment, it is expected to be better suited for clinical use. Keywords Fluorescence analysis, Vancomycin, Urine, Dosing, Therapeutic drug monitoring concentration (AUC/MIC) (Tkachuk et  al. 2018; Revilla Introduction et  al. 2010); however, trough monitoring is still a pre- Vancomycin (VCM), with a broad antimicrobial spec- ferred suitable indicator for the adjustment of VCM trum, is used as a first-line drug against methicillin- dose because of easy measurement (Liu et al. 2011; Kul- resistant Staphylococcus aureus (MRSA) (Cafferkey et al. lar et  al. 2011). Subsequent studies have reported about 1982). However, overdose of VCM may cause nephro- trough monitoring not being an ideal substitute predic- toxicity and acoustic disturbance (Jeffres et  al. 2007). tor, and the latest guidelines recommend AUC/MIC for Therefore, VCM is recommended for therapeutic drug guiding VCM dosing regimens (Rybak et  al. 2020; Mohr monitoring (TDM), and its dosage is adjusted based on and Murray 2007). Accurate determination of AUC its blood concentration (Patel et al. 2011). requires collecting blood samples numerous times from Based on pharmacokinetic/pharmacodynamic analy- patients and measuring concentrations over time, which sis, the most suitable predictor for the efficacy and safety is burdensome on patients and healthcare profession- of VCM is area under the curve/minimum inhibitory als. Therefore, there is a strong demand for a conveni - ent non-invasive or less invasive technique for collecting samples and obtaining necessary information for guiding *Correspondence: Masaru Kato VCM dosing regimens. masaru-kato@umin.ac.jp When VCM is administered intravenously, almost all Devision of Bioanalytical Chemistry, Graduate School of Pharmacy, of it is rapidly excreted in urine as an unaltered drug Showa University, 1-5-8 Hatanodai, Shinagawa-Ku, Tokyo 142-8555, Japan Department of Pharmaceutical Sciences, School of Pharmacy, Showa (Moellering et  al. 1981). Therefore, many studies have University, 1-5-8 Hatanodai, Shinagawa-Ku, Tokyo 142-8555, Japan been reported measuring VCM concentrations in the © The Author(s) 2023. 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AAPS Open (2023) 9:2 Page 2 of 8 urine instead of blood (Shokouhi et al. 2017; Baranow- Urine sample analysis ska et  al. 2009, 2010; Cass et  al. 2011; Javorska et  al. Urine was obtained from healthy five male volunteers 2017). Thus, VCM can be measured easily and fre - with ages from 20 to 50  s (median age, 41). Approxi- quently in urine instead of blood for guiding VCM dos- mately 50 mL voluntary urine was collected and centri- ing regimens. This will also aid in obtaining accurate fuged by centrifugate (MX-300, Tomy Seiko Co., Ltd., AUC values, which may be useful for designing more Tokyo, Japan) at 600 × g for 10 min to remove the cells precise dosing regimens. The advantages of using urine (Kato et  al. 2020). The supernatant of the urine was as a sample over blood are (1) self-collection by the stocked in a refrigerator. A concentration of 10 mg/mL patient; (2) collection in large quantities (1.5 L/day); (3) VCM was prepared in water. The VCM solution was urine is usually discarded and there is almost no addi- added to the stocked urine just before the measure- tional burden on the patient; (4) using a urinary cathe- ment. MonoSpin Ph (GL Sciences Inc., Tokyo, Japan) ter, it is possible to collect all urine excreted in a certain was preconditioned by centrifugate (7780II, KUBOTA period of time; and (5) there is less chance of contami- Corporation, Tokyo, Japan) at 10,000 × g for 1  min nation because the substance filtered by the glomerulus with methanol and water as per the manufacturer’s and not reabsorbed by the renal tubules is excreted. protocol. Then, 600 µL of urine sample was added to Various reports on the measurement of urinary VCM the preconditioned MonoSpin Ph and washed four have shown a good correlation between blood and urine times with 550 µL phosphate-buffered saline (PBS). concentrations (Shokouhi et  al. 2017), and urine may The captured VCM was eluted in 50 µL eluent (citrate serve as an alternative sample for guiding VCM dos- buffer (pH 2): methanol; 90:10). To the eluted solution, ing regimens. Additionally, VCM concentration in 24-h 270 µL DMSO was added, followed by 300 µL of the urine samples has been reported to be much higher than mixture solution was moved to a 96-well plate for fluo- the blood VCM concentration (several hundred µg/mL rescence analysis. to 1  mg/mL) (Javorska et  al. 2017). For measuring the concentration of VCM in blood, immunoassay is fre- HPLC measurement quently used clinically (Sattur et al. 2000; Vila et al. 2007), HPLC analyses were performed using a Hitachi and recently, a method using high performance liquid LaChrom Ultra series (Hitachi High-Tech Science chromatography (HPLC) has also been developed which Corporation, Tokyo, Japan) consisting of two L-2160 is used for short-time analysis in clinical use (Farin et al. U LaChrom Ultra pumps, an L-2200U LaChrom auto 1998; Trujillo et al. 1999; Javorska et al. 2016). However, sampler, an L-2455U LaChrom diode array detector, these analytical methods require expensive equipment, and an HPLC system organizer. A Resolve of 5-µm and it is difficult to measure high concentrations of VCM Spherical C18 column (3.9 mm × 150 mm, Waters, (several 100  mg/mL or more) accurately using common Milford, MA, USA) was used. Mobile phase A had 20 competitive immunoassays. Therefore, in this study, we mM formic acid/ammonium formate and mobile phase developed a new method for measuring urinary VCM B had 100% methanol. The gradient elution program that can be easily used in a clinical setup and can meas- of the mobile phases was as follows: 5–10% (B) from ure a wide range of VCM concentrations. 0 to 5 min. Flow rate was 1 mL/min. The injection volume was 10 µL, and a fluorescence detector with an excitation wavelength (ex) at 290 nm and emission Methods and materials wavelength (em) of 330 nm was used for detection. All Chemicals and reagents samples were filtered using a Millex-LG syringe filter Ammonium formate, citric acid anhydrous, N,N-dimeth- (pore size, 0.2 µm, Millipore) before analysis. ylformamide (DMF), dimethyl sulfoxide (DMSO), etha- nol, formic acid, methanol, trisodium citrate dihydrate, Measurement of three‑dimensional (3D) fluorescence and VCM were purchased from Fujifilm Wako Pure spectrum Chemical Corporation, Ltd. (Osaka, Japan). Glycerol The 3D fluorescence spectra were measured using was purchased from Nacalai Tesque, Inc. (Kyoto, Japan). a fluorescence spectrometer (F7100, Hitachi High Sulbactam sodium/ampicillin sodium and piperacil- Technologies Corporation, Tokyo, Japan). Excitation lin/tazobactam were obtained from Meiji Seika Pharma spectra were recorded in the 200–500  nm range with Co., Ltd. (Tokyo, Japan). Meropenem and ceftriaxone emission measurements from 200 to 500  nm. The slit were obtained from NIPRO Corporation (Osaka, Japan). width was 5  nm. The 3D spectra were shown using Cefepime was obtained from Sandoz K.K. (Tokyo, Japan). SpectAlyze software package (Dynacom Co., Ltd., Chiba, Japan). Oshima  et al. AAPS Open (2023) 9:2 Page 3 of 8 Measurement of fluorescence spectrum and intensity VCM has fluorescence intensity in the ultraviolet region The fluorescence of VCM was measured using a mul - at an excitation of 280–300  nm and emission of 290– tiplate reader (SH-9000, Corona Electric Co., Iba- 340 nm. Next, when the 3D-fluorescence spectrum of the raki, Japan). Fluorescence spectrum was obtained in urine of a healthy volunteer was measured, there were the emission wavelength from 300 to 400  nm when two fluorescence peaks, the first with an excitation of excited at 290 nm. Fluorescence intensity was obtained 250–290  nm and emission of 325–400  nm, and the sec- in the emission wavelength of 340  nm when excited at ond with an excitation of 310–360  nm and emission of 290 nm. 360–400  nm (Supporting Fig.  1). For accurate measure- ments of VCM concentration in the urine for clinical use, Measurement of VCM in a clinical laboratory it is necessary to remove fluorescent substances (amino The clinical laboratory used Emit 2000 Vancomycin acids, flavins, NADH, and others) present in urine Assay kit (Siemens Healthineers, Erlangen, Germany) (Kušnír et al. 2005) because the first fluorescence peak in and BioMajesty JCA-BM8000 series (JEOL Ltd., Tokyo, the urine sample partially overlaps with the fluorescence Japan) for the analysis. The measurements were per - of VCM. formed as described in the manufacturer’s protocol. Hence, we first determined the purification conditions for VCM from urine using a solid phase extraction (SPE) Results and discussion cartridge. A phenyl group-modified SPE cartridge was Fluorescence spectroscopy is a very sensitive and selec- selected because VCM has five aromatic rings. VCM was tive technique. Therefore, it is an ideal method for the applied to the cartridge, and each fraction was eluted in detection of trace substances in biological fluids. In PBS solution and citrate buffer (pH 2) containing 10, 15, addition, simple and inexpensive fluorometers are now 20, and 60% methanol. The eluted samples were analyzed available in the market. Supporting Fig.  1 shows the 3D by HPLC. VCM was not detected in the flow-through fluorescence spectrum of VCM at the excitation (ex.) fractions, indicating that VCM was efficiently captured wavelength range of 200–500  nm and emission (em.) in the SPE cartridge. Also, in the eluted fractions, VCM wavelength range of 200–500 nm. Warm colors (Ex: red) was detected at 9 min only in the eluted fraction of 10% indicate a strong fluorescence intensity, whereas cold methanol solution, and not in the other fractions (Fig. 1). colors (Ex: blue) indicate a weak fluorescence intensity. Specifically, urinary VCM was purified by passing the Fig. 1 Chromatograms of fractionates and 100 μg/mL VCM solutions. Column: Resolve 5-µm spherical C18 column (3.9 mm × 150 mm), mobile phase A: 20-mM formic acid/ammonium formate, B: 100% methanol, gradient program: 5–10% (B) from 0 to 5 min, flow rate: 1 mL/min, detection: fluorometry (ex/em. 290/330 nm) Oshima et al. AAPS Open (2023) 9:2 Page 4 of 8 Fig. 2 3D fluorescence spectra of washed solution. Excitation: 240–380 nm, emission 260–400 nm; the red circle indicates region of VCM fluorescence signal urine sample through an SPE cartridge, washing the car- washings (Fig.  2). This signal was derived from solvent- tridge repeatedly with PBS, and performing final elution derived Raman scattered light. in a 10% methanol solution. Then, we examined solvents that reduced the influence Next, the number of washings with PBS required for of the Raman scattering of the solvent itself. Fluorescence purification of the urine sample was examined. A urine spectra of five solvents (DMF, DMSO, methanol, glycerin, sample was placed on a cartridge, washed repeatedly and ethanol) that were excited at 290  nm are shown in with PBS, and the 3D fluorescence spectrum of the eluted Supporting Fig.  2. The scattered light of DMSO was the fractions were measured (Fig. 2). In the first washed frac - smallest among them. tion, strong fluorescence was detected near excitation of In order to completely eliminate the scattered light of 290  nm and emission of 330  nm (red circle). This indi - the eluent, the eluent must be completely evaporated and cates the elution of endogenous substances that exhibit redissolved in DMSO before fluorescence analysis. How - similar fluorescence properties to those of VCM in ever, drying of 300 μL of 10% methanol aqueous solution this fraction. The fluorescence in the region decreased is unsuitable for clinical use due to the extra time and with repeated washing, and no significant change was equipment required. Furthermore, there is a possibil- observed even after 4 or more washes. However, fluores - ity that the decomposition of VCM, which is unstable cence signal around 300–340  nm was observed and the in the solution, may occur in the drying process (White intensity remained constant regardless of the number of et  al. 1988; Serri et  al. 2017; Cao et  al. 2018). Therefore, Oshima  et al. AAPS Open (2023) 9:2 Page 5 of 8 we thought that if the volume of the eluent from the was approximately 80%, the fluorescence intensity was 10 cartridge was reduced as much as possible, and DMSO times stronger than that of the sample without DMSO, was added without drying, it would be easier to use in and even if the DMSO proportion was increased further, the clinical setup. The volume of the cartridge of SPE is the increase in the fluorescence intensity was not very approximately 20.8  μL (2.1 × 2.1 × π × 1.5 mm ) and its high. Based on the above results, we determined the pro- porosity is 80%; therefore, the void volume of the car- cedure of the analysis of urinary VCM as follows: urine tridge is 16.6 µL. In other words, it was expected that sample was centrifuged for removing cells and debris. most of the VCM captured on the cartridge would be Then, 600 µL supernatant was applied to Ph-modified eluted using an eluent several times as large as 16.6 µL. SPE cartridge and washed four times with 550 µL PBS. Hence, we loaded the cartridge with the same amount The captured VCM was eluted in 50 µL of 10% methanol, (90 µg) of VCM and measured the fluorescence of VCM and then, 270 µL of DMSO was added. The fluorescence in each fraction that was eluted in different volumes intensity (excitation at 290  nm, emission at 340  nm) (10–300 µL) of the eluent (Fig.  3a). As a result, it was of 300 µL of the mixture solution was measured by a found that some VCM was eluted even in 10 µL of elu- fluorometer. ent, and the amount of eluted VCM increased as the vol- First, we made a calibration curve in the range of ume of eluent increased. Approximately 95% of the VCM 0–50  µg/mL, which is necessary for measuring the was eluted when 300 μL eluent was used and only 36.1% therapeutic concentration range of VCM in the blood VCM was eluted by 50 μL eluent. However, their variabil- (Fig. 4). The samples were used where VCM was added ity was 3.10 and 1.51%, respectively, indicating quantita- to PBS or urine from healthy volunteers. As a result, tive elution even with a small amount of eluent. the calibration curves for urine and PBS were similar, Then, the effect of the mixing ratio of 10% metha - suggesting that contaminants in urine did not interfere nol solution, which is the eluent, and DMSO on the with the measurement of VCM. The correlation coeffi - fluorescence intensity of VCM was investigated. The cient of the calibration curve of urine and PBS was 0.94 fluorescence spectra of VCM were measured when the and 0.94, respectively. The validation data (intra-day proportion of DMSO in the measurement solvent was and inter-day reproducibility at three different concen - changed in the range of 0–97% (Fig.  3b). Even with- trations (10, 20, and 30  μg/mL) are shown in Table  1. out the DMSO solution, the fluorescence intensity The accuracy of 10, 20, and 30 μg/mL VCM solutions increased slightly by the addition of VCM. The intensity were between 93.18 and 109.76%. The RSD of intra-day was increased by increasing the proportion of DMSO and inter-day analysis were less than 6.25% and 6.28%, and it reached the maximum when the DMSO propor- respectively. Therefore, it was concluded that this assay tion was 97%. However, when the proportion of DMSO was applicable for quantitative analysis. To examine Fig. 3 a The relationship between elution volume and quantity of VCM, b relationship between ratio of DMSO to 10% methanol solution and quantity of VCM. Fluorescence spectra were obtained when excited at 290 nm. The sample volume was adjusted to 300 μL by adding 10% methanol solution before the fluorescence analysis Oshima et al. AAPS Open (2023) 9:2 Page 6 of 8 during the measurement, because it was reported that VCM is unstable in aqueous solutions (White et  al. 1988; Serri et  al. 2017; Cao et  al. 2018). Supporting Fig.  4 shows chromatograms of VCM samples immedi- ately after preparation and samples left at 37 ºC for 1 h that was more severe than the measurement conditions of 20 ºC for 30  min. The peak intensity of VCM did not change and no new peak derived from degradants appeared due to the storage. Hence, the VCM degrada- tion was neglectable during the measurement. Furthermore, since it has been reported that more than 100  µg/mL of VCM exists in urine, a calibration curve was also made using urine samples adjusted to VCM concentrations of 100, 300, 500, and 1000  µg/ mL (Vila et  al. 2007). A good calibration curve (corre- lation coefficient 0.97) was obtained even in the high- concentration region (Supporting Fig.  5). To confirm Fig. 4 Calibration curve for VCM in PBS and urine solution with the reliability of the developed method, we compared VCM concentrations from 0 to 50 μg/mL. Square and cross indicates PBS and urine, respectively. Error bars represent CV of three the measured values of the same samples with that by measurements the method currently used in clinical practice. Figure 5 shows the measurements of six different urine sam - ples using the developed method and EMIT method the effect of co-administrated drugs for the analy - by a clinical laboratory (Chen et  al. 2020). All samples sis, fluorescence of 5 frequently administrated drugs showed similar results, indicating reliability of the (sulbactam sodium/ampicillin sodium, piperacillin/ developed method for the analysis of urinary VCM. tazobactam, meropenem, ceftriaxone, and cefepime) Although urine from healthy subjects were used in was examined (Raverdy et  al. 2013). Supporting Fig.  3 this study, in order to use this method in clinical prac- shows the 3D fluorescence spectra of the 200  mg/mL tice, it is necessary to verify its effectiveness using co-administrated drug solutions. Strong fluorescence patient urine. The composition of patient specimens signal was observed from sulbactam sodium/ampicil- may differ significantly from those of healthy individu - lin sodium, and weak signal was observed from mero- als. One advantage of the developed method is that it is penem. Other co-administered drugs did not show a non-destructive method. In other words, if the meas- fluorescence signals in the range of 200 to 600  nm ured value by the developed method is abnormal, it can when excited from 200 to 600 nm. Although sulbactam be re-measured by other existing methods to confirm sodium/ampicillin sodium and meropenem showed the accuracy of the value. Therefore, we will measure fluorescence signals, the wavelength of fluorescence patient specimens using this method and obtain the signals were different from that of VCM. Hence, these AUC/MIC necessary for VCM administration design in co-administered drugs did not interfere with the VCM our next study. measurement. The degradation of VCM was evaluated Table 1. Inter-day and intra-day accuracy and validation at three different concentrations Oshima  et al. AAPS Open (2023) 9:2 Page 7 of 8 Fig. 5 Comparison of measurement values by the developed method and a clinical laboratory using EMIT method. EMIT method used Emit 2000 Vancomycin Assay kit and BioMajesty JCA-BM8000 series Funding Conclusions This work was supported by the JSPS KAKENHI. In this study, we developed a simple and rapid method for the analysis of urinary VCM. Since the method uses a Availability of data and materials Not applicable. centrifuge and fluorometer as equipment, it is cost-effec - tive compared to the other existing methods and is suit- Declarations able for clinical use. The developed method was able to measure up to 1000  μg/mL VCM, which is the assumed Ethics approval and consent to participate amount in urine samples, in contrast to the one currently All clinical studies involving human urine samples were conducted in adher- ence to the procedure approved by the Human Ethics Committee of the used in clinical laboratories (100  μg/mL). In the future, Showa University (approval no. 315), and informed consent was received from the use of this method to measure VCM in urine samples the volunteers. could help us clarify the relationship between urinary Competing interests VCM concentration and AUC/MIC and also guide VCM The authors declare that they have no competing interests. dosing regimens. Acknowledgements Received: 8 October 2022 Accepted: 9 January 2023 We acknowledge Prof. T. Sasaki, Prof. Y. Niki, Prof. I. Tokimatsu, Dr. T. Takuma, Dr. K. Karasawa, Dr. S. Murayama, Dr. Y. Naito, Mrs. K. Nakane, Dr. Y. Odanaka, and Mr. S. 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Journal

AAPS Open – Springer Journals

Published: Feb 6, 2023

Keywords: Fluorescence analysis; Vancomycin; Urine; Dosing; Therapeutic drug monitoring

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