Research article Received: 29 November 2014,
Revised: 4 February 2015,
Accepted: 14 February 2015
Published online in Wiley Online Library: 25 March 2015
(wileyonlinelibrary.com) DOI 10.1002/bio.2901
A very simple high-performance liquid chromatographic method with fluorescence detection for the determination of gemifloxacin in human breast milk Olcay Sagirli,* Seda Demirci and Armağan Önal ABSTRACT: A high-performance liquid chromatographic method with fluorescence detection was developed and validated for the determination of gemifloxacin in human breast milk. The proposed method allows the determination of gemifloxacin in breast milk samples without complex sample preparation. The samples were mixed with a mobile phase and filtered with a 0.45 μm polytetrafluoroethylene filter before analysis. Chromatographic separation was carried out on a C18 column (150 × 4.6 mm, 5 μm I.D.) using methanol:50 mM ortho-phosphoric acid solution (40:60) as the mobile phase with a 1.0 mL/min flow rate. Quantitation was performed using fluorescence detection with an excitation wavelength at 272 nm and an emission wavelength at 395 nm. The linear range was found to be 0.1–2.5 μg/mL. The method was applied successfully for the determination of gemifloxacin in breast milk obtained from a breastfeeding mother after oral administration of a single tablet that included 320 mg gemifloxacin per gemifloxacin tablet. Copyright © 2015 John Wiley & Sons, Ltd. Keywords: gemifloxacin; liquid chromatography; fluorescence detection; human breast milk; validation
1326
Introduction
Experimental
The World Health Organization (WHO) recommends breastfeeding exclusively during the first 6 months of life (1). However, infants of breastfeeding mothers undergoing drug therapy could be exposed to the adverse effects or toxicity transition of the mother’s medication into the breast milk. For this reason, it is important to determine the extent of drug concentration excreted into breast milk. Gemifloxacin (GEM), 7-[(4Z)-3-(aminomethyl)-4-methoxyiminopyrrolidin-1-yl]-1-cyclopropyl-6-fluoro-4-oxo-1,8-naphthyridine-3carboxylic acid is a new fluoroquinolone antibacterial compound (Fig. 1) that is used for the treatment of respiratory problems, acute bacterial exacerbation of chronic bronchitis, community-acquired pneumonia, and urinary tract infections (2–5). Several reports in the literature have detailed the analysis of GEM by numerous methods using different biological matrices by high-performance liquid chromatography (HPLC) and UV light (6,7), fluorescence (8–10) or tandem mass spectrometry (MS/MS) (11,12) detection. To our knowledge, however, there is currently no available method for determining GEM in breast milk samples. The aim of this study was to determine the concentration of GEM in breast milk samples using a HPLC-fluorescence detection method without extraction. Before analysis, samples were diluted with a mobile phase and filtered with a 0.45 μm polytetrafluoroethylene (PTFE) filter. No interference was observed from the milk matrices and good reproducible results were obtained. The developed method was validated and applied successfully to determine the concentration of GEM excreted into breast milk samples.
Material and chemicals
Luminescence 2015; 30: 1326–1329
Gemifloxacin mesylate (in its pharmaceutical preparation – Factive Film Tablet®; 320 mg GEM) was kindly provided by Abdi Ibrahim Pharmaceuticals (Istanbul, Turkey). All chemicals and solvents were of analytical and gradient grade. HPLC grade ultra-pure water was prepared by the ELGA Purelab Option-Q DV 25 (UK) water purification system. Sartorius minisart SRP25 PTFE 0.45 μm filters were used for sample filtration. Instrumentation and chromatographic conditions Chromatographic separations were performed on a Shimadzu LC 20A (Kyoto, Japan) liquid chromatograph system, consisting of a model LC 20 AT solvent delivery system, a SIL-20AC autosampler system and using a model CTO 20A column oven. The RF 10 AXL fluorescence detector was set to an excitation wavelength of 272 nm and an emission wavelength of 395 nm. Detector gain was set at 4 and the sensitivity was set at medium. Separation was performed on a Thermo Hypersil Gold C18 column (150 mm × 4.6 mm, 5 μm I.D.) with a guard column (4 mm × 3 mm, 5 μm I.D.) packed with the same material. * Correspondence to: O. Sagirli, Istanbul University, Faculty of Pharmacy, Department of Analytical Chemistry, 34116, Beyazit, Istanbul–Turkey. Tel: +90 2124400000 13587; Fax: +90 2124400252. E-mail: olcaysagirli@ yahoo.com Istanbul University Faculty of Pharmacy, Department of Analytical Chemistry, 34116, Beyazit, Istanbul, Turkey
Copyright © 2015 John Wiley & Sons, Ltd.
Determination of Gemifloxacin in Breast Milk by HPLC
Figure 1. Chemical structure of gemifloxacin mesylate.
Isocratic elution was carried out with methanol:50 mM orthophosphoric acid solution (40:60). The flow rate of the mobile phase was 1.0 mL/min, the temperature of the column was held at 30°C. The data were collected and analysed via the LC Solution (Version 1.24) system software.
Preparation of standard solutions The stock solution of GEM was prepared by dissolving an amount corresponding to 13.33 mg mg/mL concentration of GEM mesylate (equivalent to 10 mg GEM base) in water and then diluted with water to give a 1 mg/mL concentration. Standard solutions of GEM were prepared by dilution of the stock solutions with the mobile phase. The stock solutions were stored at 4°C and were stable for 1 month.
Sample preparation Drug-free human breast milk samples were obtained from a normal healthy informed volunteer (A. Önal, one of the researchers in this study, 37 years old, 70 kg, 178 cm in height). Breast milk (1 mL) was spiked with GEM solutions to give a final concentration of each drug of 0.1–2.5 μg/mL, then diluted with mobile phase to 5 mL and vortexed for 1 min. Each sample was filtered with a 0.45 μm PTFE filter and 20 μL of filtrate were injected into the HPLC system.
Method validation The developed analytical method was validated according to international guidelines with respect to certain parameters such as selectivity, accuracy, precision, recovery, calibration curve, sensitivity, reproducibility and stability of analyte in spiked samples (13). Breast milk calibration standards were prepared at six levels by spiking 1 mL of human breast milk samples with 0.1–2.5 μg/mL concentrations of GEM. The obtained mixture was subjected to the protocol described previously in the Sample preparation
section and injected into the HPLC system. Calibration curves were set up by the least-square method using the drug peak areas against the corresponding concentrations of the analyte (as μg/mL). Linear regression analysis of the data from calibration curves gave slope (a), intercept (b) and coefficient of determination, which were used to determine the concentration of analyte in the quality control (QC) samples. The lowest concentration level that gave a chromatographic response with acceptable coefficient of variation was defined as the limit of quantitation (LOQ). Method precision and accuracy were determined in terms of repeatability (intra-day precision) and intermediate precision (inter-day precision). In order to determine precision and accuracy, QC milk samples spiked at three different concentration levels (0.1, 1.0 and 2.5 μg/mL) of GEM were analysed six times a day in triplicate injections over 6 consecutive days in 2 months. The assay accuracy was calculated as relative mean error (RME). The measurement of precision was given as the relative standard deviation (RSD). Accuracy and precision values within ±15% covering the actual range of experimental concentrations were considered acceptable. The breast milk samples were treated as noted in the Sample preparation section, and injected into the HPLC system in triplicate and expressed as mean ± standard deviation (SD) and RSD calculated from data obtained. Drug stability in breast milk samples was assessed using low (0.1 μg/mL) and high (2.5 μg/mL) QC samples under different conditions in the autosampler at room temperature for 8 h, after three freeze–thaw cycles, on storage at 20 °C for 14 days, and in the autosampler after processing for 8 h. For the GEM stability studies with the mobile phase, samples were kept in the dark at ambient temperature and at +4°C for 12, 24, 48, 72 or 96 h and analysed in the HPLC system. Application to human breast milk samples The developed method was used to investigate the breast milk profile of GEM in a healthy volunteer (37 years old, 70 kg, 178 cm height) after administrating a single oral dose of 320 mg GEM. The lactating volunteer was enrolled on the basis that she was to stop breastfeeding at the time of the study. Breast milk was collected at the third hour following oral drug administration, because the maximum blood-plasma concentration (Cmax) is achieved at the third hour following oral administration. The possible excretion of active ingredients into the breast milk is expected to be at its highest level after a single oral dose oral and provided the volunteer sufficient time to regenerate milk in the breasts. Both breasts were emptied of milk by breast pump and the exact times of starting and finishing expression were
Luminescence 2015; 30: 1326–1329
Copyright © 2015 John Wiley & Sons, Ltd.
wileyonlinelibrary.com/journal/luminescence
1327
Figure 2. Chromatograms of (A) blank milk sample; (B) breast milk sample spiked with GEM (2 μg/mL); (C) breast milk sample obtained from a healthy volunteer 3 h after oral administration of 320 mg GEM.
O. Sagirli et al. recorded. The total volume of milk sample was recorded and 10 mL aliquots were taken for analysis. The mixing process was used to ensure that the GEM concentration was representative of infant exposure across a feeding period. Any excess milk collected at expression was discarded. The breast milk samples were stored at 20 °C until analysis.
Table 2. Intra-day precision Added concentration (μg/mL) 0.1 Found concentration (μg/mL)
Results and discussion Chromatographic conditions Different types of analytical columns were tested including C8, CN and C18. Trials showed that the C18 column gave symmetrical and sharp peaks. Acidic and aqueous mobile phases were tested, and acidic mobile phase provided good results; ortho-phosphoric acid solution (50 mM) was preferred as the acidic solution. A mobile phase composition of methanol:50 mM ortho-phosphoric acid solution (40:60) with a flow rate of 1.0 mL/min was used to achieve a good resolution. For quantitative analytical purposes, an excitation wavelength at 272 nm and an emission wavelength at 395 nm using fluorescence detection were chosen. The retention time of the GEM obtained under these conditions was 4.4 min (Fig. 2B).
Mean SD* RSD* (%) RME* (%)
1.0
0.0995 0.0946 0.0994 0.0992 0.0987 0.0990 0.0984 0.0019 1.91 1.60
0.998 0.990 0.960 0.982 0.993 1.005 0.988 0.016 1.59 1.20
2.5 2.484 2.425 2.434 2.498 2.492 2.490 2.471 0.032 1.30 1.18
* SD: standard deviation, RSD: relative standard deviation, RME: relative mean error.
Table 3. Inter-day precision Method validation The calibration graphs were constructed according to the pharmacokinetic data (7) of GEM obtained from the human plasma study. The GEM peak areas obtained were plotted against the corresponding concentrations of GEM (as μg/mL) and the calibration curves were set up at a concentration range of 0.1–2.5 μg/mL in breast milk. The mean linear regression equation of the calibration curves was y ¼ 124225:49x – 4199:02 r 2 ¼ 0:9992 ; where y represents the GEM peak area and x represents GEM concentrations in the breast milk. The coefficient of determination values of the calibration curves ranged from 0.9950–0.9995, which showed the good linearity of the method. The limit of detection (LOD) and LOQ were determined under described conditions. The LOQ value for each sample was accepted as the lowest concentration on the calibration curve as 0.1 μg/mL. Under the experimental conditions, the LOD value was 0.03 μg/mL at an S/N ratio of 3. Blank human breast milk samples were spiked with analytes at three different GEM concentration levels (0.1, 1.0 or 2.5 μg/mL). The mean recoveries of GEM extracted from breast milk samples were found to be 98.85 %. Results obtained are presented in Table 1. The intra- and inter-day precisions were examined by analysis of drugs for same day and on 7 consecutive days (each n = 5). The Table 1. Extraction recovery of GEM from breast milk samples Added concentration (μg/mL)
Found concentration (μg/mL) ± SD*
Recovery %
0.1 1.0 2.5
0.0985 ± 0.0019 0.988 ± 0.012 2.481 ± 0.018
98.50 98.80 99.24
0.1 Found concentration (μg/mL)
Mean SD* RSD* (%) RME* (%)
0.0989 0.0995 0.0962 0.0991 0.0997 0.0950 0.0981 0.002 2.01 1.93
1.0 0.980 0.960 1.010 0.990 0.975 0.985 0.983 0.017 1.69 1.67
2.5 2.460 2.455 2.447 2.494 2.475 2.435 2.461 0.021 0.85 1.56
* SD: standard deviation, RSD: relative standard deviation, RME: relative mean error.
RSD values for intra-day precision 1.30–1.91% (Table 2) and for the inter-day precision 0.85–2.01% (Table 3) were found for all developed methods, which indicated good precision. The stabilities of drug standard solutions were tested under several storage conditions (room temperature in the dark for 24 h, autosampler conditions for 24 h, and at 4°C for 1 month). Stability studies indicated that the samples were stable when kept at room temperature for 24 h, autosampler conditions for 24 h and refrigerated at 4°C for 1 month. Under all conditions tested, GEM was found to be stable.
RSD* Application to human breast milk samples 1.93 1.22 0.73
1328
* SD: standard deviation; RSD: relative standard deviation.
wileyonlinelibrary.com/journal/luminescence
Added concentration (μg/mL)
The proposed method was applied to the determination of GEM in human breast milk samples. The chromatogram of a breast milk sample from a 37-year-old healthy volunteer who took a single dose of Factive tablet (containing 320 mg GEM) is shown in Fig. 2(C).
Copyright © 2015 John Wiley & Sons, Ltd.
Luminescence 2015; 30: 1326–1329
Determination of Gemifloxacin in Breast Milk by HPLC
Conclusions A very simple, rapid, specific HPLC method based on fluorescence detection was developed for the determination of GEM in human breast milk samples. The important advantage of the proposed method is the simplicity of the sample clean-up procedure. The samples were diluted with a mobile phase and filtered just prior to analysis. The method has a short elution time (4.4 min) and a low LOD (0.03 μg/mL) value for GEM. The developed and validated method was applied successfully to the analysis of GEM in breast milk samples from a 320 mg single dose treatment of a 37-year-old healthy volunteer. Results shows that the method is suitable for the determination of GEM concentrations in human breast milk samples.
References 1. World Health Organization, UNICEF. Global strategy for infant and young child feeding 2003. Geneva. http://whqlibdoc.who.int/publications/2003/9241562218.pdf [cited 16 May 2012]. 2. Völgyi G, Vizserálek G, Takács-Novák K, Avdeef A Tam KY. Predicting the exposure and antibacterial activity of fluoroquinolones based on physicochemical properties. Eur J Pharm Sci 2012;47:21–7. 3. Oh JI, Paek KS, Ahn MJ, Kim MY, Hong CY, Kim IC, et al. In vitro and in vivo evaluations of LB20304, a new fluoronaphthyridone. Antimicrob Agents Chemother 1996;40:1564–8. 4. Vamsi Krishna M, Sanker DG. Utility of σ and π-acceptors for the spectrophotometric determination of gemifloxacin mesylate in pharmaceutical formulations. Hindawi J Chem 2008;5:493–8.
5. Marrie TJ. Community-acquired pneumonia: epidemiology, etiology, treatment. Infect Dis Clin North Am 1998;12:723–40. 6. Rote AR, Pingle SP. Reverse phase-HPLC and HPTLC methods for determination of gemifloxacin mesylate in human plasma. J Chromatogr B 2009;877:3719–23. 7. Chakrabarty US, Das A, Bhaumik U, Chatterjee B, Ghosh A, Bose A, et al. Rapid and sensitive LC method for the analysis of gemifloxacin in human plasma. Chromatographia 2009;69:853. 8. Kaiser M, Grünspan LD, Costa TD Tasso L. Reversed phase liquid chromatography method with fluorescence detection of gemifloxacin in rat plasma and its application to the pharmacokinetic study. J Chromatogr B 2011;879:3639–44. 9. de Araújo BV, Laureano JV, Grünspan LD, Dalla CT Tasso L. Validation of an efficient LC-microdialysis method for gemifloxacin quantitation in lung, kidney and liver of rats. J Chromatogr B 2011;919–920:62–6. 10. Nageswara RR, Naidu CG, Guru PK, Padiya R Agwane SB. Determination of gemifloxacin on dried blood spots by hydrophilic interaction liquid chromatography with fluorescence detector: application to pharmacokinetics in rats. Biomed Chromatogr 2012;26:1534–42. 11. Rodríguez RV, Smyth WF. A study of the analytical behaviour of selected new molecular entities using electrospray ionisation ion trap mass spectrometry, liquid chromatography, gas chromatography and polarography and their determination in serum at therapeutic concentrations. Anal Chim Acta 2008;623:221–30. 12. Kadi AA, Angawi RF, Attwa MW, Darwish HW, Abdelhameed AS. High throughput quantitative bioanalytical LC/MS/MS determination of gemifloxacin in human urine. J Chem 2013;2013:9. doi: 10.1155/2013/ 905704. 13. US Food and Drug Administration. Guidance for industry bioanalytical method validation. Accessed on 17 July 2014, Available from: http:// www.fda.gov/downloads/Drugs/Guidances/ucm070107.pdf
1329
Luminescence 2015; 30: 1326–1329
Copyright © 2015 John Wiley & Sons, Ltd.
wileyonlinelibrary.com/journal/luminescence
Revised: 4 February 2015,
Accepted: 14 February 2015
Published online in Wiley Online Library: 25 March 2015
(wileyonlinelibrary.com) DOI 10.1002/bio.2901
A very simple high-performance liquid chromatographic method with fluorescence detection for the determination of gemifloxacin in human breast milk Olcay Sagirli,* Seda Demirci and Armağan Önal ABSTRACT: A high-performance liquid chromatographic method with fluorescence detection was developed and validated for the determination of gemifloxacin in human breast milk. The proposed method allows the determination of gemifloxacin in breast milk samples without complex sample preparation. The samples were mixed with a mobile phase and filtered with a 0.45 μm polytetrafluoroethylene filter before analysis. Chromatographic separation was carried out on a C18 column (150 × 4.6 mm, 5 μm I.D.) using methanol:50 mM ortho-phosphoric acid solution (40:60) as the mobile phase with a 1.0 mL/min flow rate. Quantitation was performed using fluorescence detection with an excitation wavelength at 272 nm and an emission wavelength at 395 nm. The linear range was found to be 0.1–2.5 μg/mL. The method was applied successfully for the determination of gemifloxacin in breast milk obtained from a breastfeeding mother after oral administration of a single tablet that included 320 mg gemifloxacin per gemifloxacin tablet. Copyright © 2015 John Wiley & Sons, Ltd. Keywords: gemifloxacin; liquid chromatography; fluorescence detection; human breast milk; validation
1326
Introduction
Experimental
The World Health Organization (WHO) recommends breastfeeding exclusively during the first 6 months of life (1). However, infants of breastfeeding mothers undergoing drug therapy could be exposed to the adverse effects or toxicity transition of the mother’s medication into the breast milk. For this reason, it is important to determine the extent of drug concentration excreted into breast milk. Gemifloxacin (GEM), 7-[(4Z)-3-(aminomethyl)-4-methoxyiminopyrrolidin-1-yl]-1-cyclopropyl-6-fluoro-4-oxo-1,8-naphthyridine-3carboxylic acid is a new fluoroquinolone antibacterial compound (Fig. 1) that is used for the treatment of respiratory problems, acute bacterial exacerbation of chronic bronchitis, community-acquired pneumonia, and urinary tract infections (2–5). Several reports in the literature have detailed the analysis of GEM by numerous methods using different biological matrices by high-performance liquid chromatography (HPLC) and UV light (6,7), fluorescence (8–10) or tandem mass spectrometry (MS/MS) (11,12) detection. To our knowledge, however, there is currently no available method for determining GEM in breast milk samples. The aim of this study was to determine the concentration of GEM in breast milk samples using a HPLC-fluorescence detection method without extraction. Before analysis, samples were diluted with a mobile phase and filtered with a 0.45 μm polytetrafluoroethylene (PTFE) filter. No interference was observed from the milk matrices and good reproducible results were obtained. The developed method was validated and applied successfully to determine the concentration of GEM excreted into breast milk samples.
Material and chemicals
Luminescence 2015; 30: 1326–1329
Gemifloxacin mesylate (in its pharmaceutical preparation – Factive Film Tablet®; 320 mg GEM) was kindly provided by Abdi Ibrahim Pharmaceuticals (Istanbul, Turkey). All chemicals and solvents were of analytical and gradient grade. HPLC grade ultra-pure water was prepared by the ELGA Purelab Option-Q DV 25 (UK) water purification system. Sartorius minisart SRP25 PTFE 0.45 μm filters were used for sample filtration. Instrumentation and chromatographic conditions Chromatographic separations were performed on a Shimadzu LC 20A (Kyoto, Japan) liquid chromatograph system, consisting of a model LC 20 AT solvent delivery system, a SIL-20AC autosampler system and using a model CTO 20A column oven. The RF 10 AXL fluorescence detector was set to an excitation wavelength of 272 nm and an emission wavelength of 395 nm. Detector gain was set at 4 and the sensitivity was set at medium. Separation was performed on a Thermo Hypersil Gold C18 column (150 mm × 4.6 mm, 5 μm I.D.) with a guard column (4 mm × 3 mm, 5 μm I.D.) packed with the same material. * Correspondence to: O. Sagirli, Istanbul University, Faculty of Pharmacy, Department of Analytical Chemistry, 34116, Beyazit, Istanbul–Turkey. Tel: +90 2124400000 13587; Fax: +90 2124400252. E-mail: olcaysagirli@ yahoo.com Istanbul University Faculty of Pharmacy, Department of Analytical Chemistry, 34116, Beyazit, Istanbul, Turkey
Copyright © 2015 John Wiley & Sons, Ltd.
Determination of Gemifloxacin in Breast Milk by HPLC
Figure 1. Chemical structure of gemifloxacin mesylate.
Isocratic elution was carried out with methanol:50 mM orthophosphoric acid solution (40:60). The flow rate of the mobile phase was 1.0 mL/min, the temperature of the column was held at 30°C. The data were collected and analysed via the LC Solution (Version 1.24) system software.
Preparation of standard solutions The stock solution of GEM was prepared by dissolving an amount corresponding to 13.33 mg mg/mL concentration of GEM mesylate (equivalent to 10 mg GEM base) in water and then diluted with water to give a 1 mg/mL concentration. Standard solutions of GEM were prepared by dilution of the stock solutions with the mobile phase. The stock solutions were stored at 4°C and were stable for 1 month.
Sample preparation Drug-free human breast milk samples were obtained from a normal healthy informed volunteer (A. Önal, one of the researchers in this study, 37 years old, 70 kg, 178 cm in height). Breast milk (1 mL) was spiked with GEM solutions to give a final concentration of each drug of 0.1–2.5 μg/mL, then diluted with mobile phase to 5 mL and vortexed for 1 min. Each sample was filtered with a 0.45 μm PTFE filter and 20 μL of filtrate were injected into the HPLC system.
Method validation The developed analytical method was validated according to international guidelines with respect to certain parameters such as selectivity, accuracy, precision, recovery, calibration curve, sensitivity, reproducibility and stability of analyte in spiked samples (13). Breast milk calibration standards were prepared at six levels by spiking 1 mL of human breast milk samples with 0.1–2.5 μg/mL concentrations of GEM. The obtained mixture was subjected to the protocol described previously in the Sample preparation
section and injected into the HPLC system. Calibration curves were set up by the least-square method using the drug peak areas against the corresponding concentrations of the analyte (as μg/mL). Linear regression analysis of the data from calibration curves gave slope (a), intercept (b) and coefficient of determination, which were used to determine the concentration of analyte in the quality control (QC) samples. The lowest concentration level that gave a chromatographic response with acceptable coefficient of variation was defined as the limit of quantitation (LOQ). Method precision and accuracy were determined in terms of repeatability (intra-day precision) and intermediate precision (inter-day precision). In order to determine precision and accuracy, QC milk samples spiked at three different concentration levels (0.1, 1.0 and 2.5 μg/mL) of GEM were analysed six times a day in triplicate injections over 6 consecutive days in 2 months. The assay accuracy was calculated as relative mean error (RME). The measurement of precision was given as the relative standard deviation (RSD). Accuracy and precision values within ±15% covering the actual range of experimental concentrations were considered acceptable. The breast milk samples were treated as noted in the Sample preparation section, and injected into the HPLC system in triplicate and expressed as mean ± standard deviation (SD) and RSD calculated from data obtained. Drug stability in breast milk samples was assessed using low (0.1 μg/mL) and high (2.5 μg/mL) QC samples under different conditions in the autosampler at room temperature for 8 h, after three freeze–thaw cycles, on storage at 20 °C for 14 days, and in the autosampler after processing for 8 h. For the GEM stability studies with the mobile phase, samples were kept in the dark at ambient temperature and at +4°C for 12, 24, 48, 72 or 96 h and analysed in the HPLC system. Application to human breast milk samples The developed method was used to investigate the breast milk profile of GEM in a healthy volunteer (37 years old, 70 kg, 178 cm height) after administrating a single oral dose of 320 mg GEM. The lactating volunteer was enrolled on the basis that she was to stop breastfeeding at the time of the study. Breast milk was collected at the third hour following oral drug administration, because the maximum blood-plasma concentration (Cmax) is achieved at the third hour following oral administration. The possible excretion of active ingredients into the breast milk is expected to be at its highest level after a single oral dose oral and provided the volunteer sufficient time to regenerate milk in the breasts. Both breasts were emptied of milk by breast pump and the exact times of starting and finishing expression were
Luminescence 2015; 30: 1326–1329
Copyright © 2015 John Wiley & Sons, Ltd.
wileyonlinelibrary.com/journal/luminescence
1327
Figure 2. Chromatograms of (A) blank milk sample; (B) breast milk sample spiked with GEM (2 μg/mL); (C) breast milk sample obtained from a healthy volunteer 3 h after oral administration of 320 mg GEM.
O. Sagirli et al. recorded. The total volume of milk sample was recorded and 10 mL aliquots were taken for analysis. The mixing process was used to ensure that the GEM concentration was representative of infant exposure across a feeding period. Any excess milk collected at expression was discarded. The breast milk samples were stored at 20 °C until analysis.
Table 2. Intra-day precision Added concentration (μg/mL) 0.1 Found concentration (μg/mL)
Results and discussion Chromatographic conditions Different types of analytical columns were tested including C8, CN and C18. Trials showed that the C18 column gave symmetrical and sharp peaks. Acidic and aqueous mobile phases were tested, and acidic mobile phase provided good results; ortho-phosphoric acid solution (50 mM) was preferred as the acidic solution. A mobile phase composition of methanol:50 mM ortho-phosphoric acid solution (40:60) with a flow rate of 1.0 mL/min was used to achieve a good resolution. For quantitative analytical purposes, an excitation wavelength at 272 nm and an emission wavelength at 395 nm using fluorescence detection were chosen. The retention time of the GEM obtained under these conditions was 4.4 min (Fig. 2B).
Mean SD* RSD* (%) RME* (%)
1.0
0.0995 0.0946 0.0994 0.0992 0.0987 0.0990 0.0984 0.0019 1.91 1.60
0.998 0.990 0.960 0.982 0.993 1.005 0.988 0.016 1.59 1.20
2.5 2.484 2.425 2.434 2.498 2.492 2.490 2.471 0.032 1.30 1.18
* SD: standard deviation, RSD: relative standard deviation, RME: relative mean error.
Table 3. Inter-day precision Method validation The calibration graphs were constructed according to the pharmacokinetic data (7) of GEM obtained from the human plasma study. The GEM peak areas obtained were plotted against the corresponding concentrations of GEM (as μg/mL) and the calibration curves were set up at a concentration range of 0.1–2.5 μg/mL in breast milk. The mean linear regression equation of the calibration curves was y ¼ 124225:49x – 4199:02 r 2 ¼ 0:9992 ; where y represents the GEM peak area and x represents GEM concentrations in the breast milk. The coefficient of determination values of the calibration curves ranged from 0.9950–0.9995, which showed the good linearity of the method. The limit of detection (LOD) and LOQ were determined under described conditions. The LOQ value for each sample was accepted as the lowest concentration on the calibration curve as 0.1 μg/mL. Under the experimental conditions, the LOD value was 0.03 μg/mL at an S/N ratio of 3. Blank human breast milk samples were spiked with analytes at three different GEM concentration levels (0.1, 1.0 or 2.5 μg/mL). The mean recoveries of GEM extracted from breast milk samples were found to be 98.85 %. Results obtained are presented in Table 1. The intra- and inter-day precisions were examined by analysis of drugs for same day and on 7 consecutive days (each n = 5). The Table 1. Extraction recovery of GEM from breast milk samples Added concentration (μg/mL)
Found concentration (μg/mL) ± SD*
Recovery %
0.1 1.0 2.5
0.0985 ± 0.0019 0.988 ± 0.012 2.481 ± 0.018
98.50 98.80 99.24
0.1 Found concentration (μg/mL)
Mean SD* RSD* (%) RME* (%)
0.0989 0.0995 0.0962 0.0991 0.0997 0.0950 0.0981 0.002 2.01 1.93
1.0 0.980 0.960 1.010 0.990 0.975 0.985 0.983 0.017 1.69 1.67
2.5 2.460 2.455 2.447 2.494 2.475 2.435 2.461 0.021 0.85 1.56
* SD: standard deviation, RSD: relative standard deviation, RME: relative mean error.
RSD values for intra-day precision 1.30–1.91% (Table 2) and for the inter-day precision 0.85–2.01% (Table 3) were found for all developed methods, which indicated good precision. The stabilities of drug standard solutions were tested under several storage conditions (room temperature in the dark for 24 h, autosampler conditions for 24 h, and at 4°C for 1 month). Stability studies indicated that the samples were stable when kept at room temperature for 24 h, autosampler conditions for 24 h and refrigerated at 4°C for 1 month. Under all conditions tested, GEM was found to be stable.
RSD* Application to human breast milk samples 1.93 1.22 0.73
1328
* SD: standard deviation; RSD: relative standard deviation.
wileyonlinelibrary.com/journal/luminescence
Added concentration (μg/mL)
The proposed method was applied to the determination of GEM in human breast milk samples. The chromatogram of a breast milk sample from a 37-year-old healthy volunteer who took a single dose of Factive tablet (containing 320 mg GEM) is shown in Fig. 2(C).
Copyright © 2015 John Wiley & Sons, Ltd.
Luminescence 2015; 30: 1326–1329
Determination of Gemifloxacin in Breast Milk by HPLC
Conclusions A very simple, rapid, specific HPLC method based on fluorescence detection was developed for the determination of GEM in human breast milk samples. The important advantage of the proposed method is the simplicity of the sample clean-up procedure. The samples were diluted with a mobile phase and filtered just prior to analysis. The method has a short elution time (4.4 min) and a low LOD (0.03 μg/mL) value for GEM. The developed and validated method was applied successfully to the analysis of GEM in breast milk samples from a 320 mg single dose treatment of a 37-year-old healthy volunteer. Results shows that the method is suitable for the determination of GEM concentrations in human breast milk samples.
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Luminescence 2015; 30: 1326–1329
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