Exploring strategies to individualize treatment with aminoglycosides and co-trimoxazole for
MDR Tuberculosis
Dijkstra, Jacob Albert
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2017
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Dijkstra, J. A. (2017). Exploring strategies to individualize treatment with aminoglycosides and
co-trimoxazole for MDR Tuberculosis. Rijksuniversiteit Groningen.
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Quantification
of Amikacin and
Kanamycin in Serum
Using a Simple and
Validated Liquid
Chromatography-Tandem Mass
Spectrometry Method
J.A. Dijkstra M.G.G. Sturkenboom K. van Hateren R.A. Koster B. Greijdanus J.W.C. Alffenaar39
ABSTRACT
Amikacin and kanamycin are frequently used in the treatment of multidrug resistant tuberculosis. The commercially available immunoassay is unable to analyze kanamycin and through levels of amikacin. The objective was therefore to develop a LC-MS/MS method for the quantification of amikacin and kanamycin in human serum.
The presented LC-MS/MS method meets the recommendations of the FDA with a low lower limit of quantification (LLOQ) of 250 ng/mL for amikacin and 100 ng/mL for kanamycin. No statistical significant difference was found between the LC-MS/MS and the immunoassay of amikacin (Architect, P = 0.501).
The lower LLOQ of amikacin and the ability to analyze kanamycin makes the LC-MS/MS method the preferred method for analyzing these aminoglycosides.
INTRODUCTION
Tuberculosis (TB) is an infectious disease that kills almost 2 million patients every year.1 Multidrug
resistant tuberculosis (MDR-TB) is caused by strains of Mycobacterium tuberculosis resistant to at least two first line drugs: rifampin and isoniazid.1,2 Annually, 500,000 patients get infected with an
MDR-TB strain.1 The aminoglycosides amikacin and kanamycin are both frequently used for the
treatment of MDR-TB1,2, since minimal inhibitory concentrations (MIC) range from 1,000 to 4,000
ng/mL.3 However, a reliable, robust method without extensive sample pre-processing to measure
both aminoglycosides in plasma or serum is not available until now.
The effective pharmacodynamic parameter for aminoglycosides is the peak serum concentration (Cmax) over MIC.4 To reduce the emergence of resistance and to optimize the efficacy,
a Cmax/MIC ratio of ≥ 8 is preferred.3–5 Common side effects associated with aminoglycosides
therapy are ototoxicity and nephrotoxicity, both possibly related to the treatment duration and the
cumulative dose.6–8 Furthermore, a study with human subjects showed a significant correlation
between the average trough level (Cmin) of aminoglycosides and auditory toxicity.9 This effect could
be more important in patients with TB, whom are often treated for months with aminoglycosides. Although evidence is limited, monitoring and minimizing trough levels seems justifiable.
Therefore, both Cmax and Cmin are important parameters in therapeutic drug monitoring
(TDM) of aminoglycosides. TDM may help to optimize the dose if measured serum concentrations
are not within the desired therapeutic range.10 To enable TDM, a specific and sensitive method
of analysis is required. There is no commercially available immunoassay for the quantification of kanamycin. Furthermore, the immunoassay available for amikacin has a high lower limit of quantification (LLOQ) of 1500 ng/mL.
In addition, the available immunoassay is less versatile than newer methods of analysis, such as liquid chromatography tandem mass spectrometry (LC-MS/MS). The Stop TB Partnership proposed in the Global Plan to Stop TB 2011-2015 that laboratory strengthening is one of the key
components in the fight against TB.11 With one single LC-MS/MS, TDM could be performed for
many drugs, improving efficacy and reducing toxicity, thereby hopefully increasing compliance. Literature about analysis of amikacin and kanamycin in serum using LC-MS/MS is scarcely
available.12,13 A published LC-MS/MS method used solid-phase extraction (SPE) with different types
of columns, but this is a time-consuming method with runtimes up to 12 minutes.12 Furthermore,
no internal standard (IS) was used. The authors used only external calibrations.12
Baietto et al. developed a sample preparation method without SPE for LC-MS/MS.13
Unfortunately, this method was not validated for kanamycin. Moreover, this method requires an additional dilution step and used quinoxaline as IS, which is not structurally related to amikacin
40
or kanamycin.13 Therefore, both described methods are less able to compensate for sample and
matrix variability.14
A recent study described the simultaneous analysis of nine second-line anti-tuberculous drugs.15 Unfortunately, amikacin was not included in the method validation. Furthermore, the lower limit of quantification of kanamycin (LLOQ) was 2500 ng/mL and therefore relatively high.
To enable the use of TDM in the therapy with amikacin or kanamycin, a suitable, sensitive and robust method is required to analyse amikacin and kanamycin over a wide concentration range. The objective of this study is to develop and validate a simple and fast LC-MS/MS method for the quantification of amikacin and kanamycin in human serum for routine patient care and clinical studies.
MATERIALS AND METHODS
Analysis
Kanamycin and amikacin were purchased from Sigma Aldrich (St. Louis, MO, USA). Heptafluorobutyric acid anhydride (HFBAA) was obtained from Fluka (Sigma Aldrich, St. Louis, MO, USA). Water was purified with a Milli-Q system (Millipore Corporation, Billerica, MA, USA). Buffer solution consisted of 1% HFBAA in purified water. The anhydride formulation was already available in our laboratory. Trichloroacetic acid (TCA) was used in sample processing and was purchased from Merck (Whitehouse Station, NJ, USA). Eluent for liquid chromatography consisted of buffer
solution, purified water and methanol (Lichrosolv®, Merck, Whitehouse Station, NJ, USA). IS stock
solution consisted of 5000 ng/mL apramycin (Sigma Aldrich, St. Louis, MO, USA) in HFBAA (1%). To 100 μL serum, 50 μL TCA was added. In addition, 50 μL IS stock solution was added. After each addition the sample was vortexed for 1 minute. The subsequent sample was centrifuged (5 minutes at 11,000 rpm) and the supernatant was transferred into a clear glass vial with a silicone/ PTFE septum.
A tandem mass spectrometer was used to perform the analysis. The mass spectrometer was a Finnigan TSQ Quantum Discovery Max (TSQ Quantum, Thermo Fisher, San Jose, CA, USA), supplied with a Finnigan Surveyor MS Pump Plus and a Finnigan Autosampler Plus. Positive electron spray ionization was performed at 3500V. The liquid chromatography (LC) system was equipped with a Thermo Scientific Hypurity C18, 5.0 * 2.1 mm column with a particle size of 3 μm. Separation took place with gradient elution and a flow of 300 μL/min at approximately 100 bar. The gradient elution used is shown in table 1.
The autosampler was configured to an injection volume of 5 μL and a tray temperature of 10 °C. Nitrogen was used as sheath and auxiliary gas and argon was used as collision gas. Sheath gas pressure was set to 35 arbitrary units, auxiliary gas to 5 arbitrary units. Used mass transitions are shown in table 2.
Table 1. Gradient elution Minutes HFBAA
(1% in water) Water Methanol
0 5% 77.5% 17.5%
0.50 5% 50% 45%
1.20 5% 43% 52%
5.51 5% 77.5% 17.5%
6.00 5% 77.5% 17.5%
Table 2. MS/MS conditions and mass transitions Compound Tube lens
(V) Collision energy (eV) Mass transition (m/z) Amikacin 109 33 586.2 → 163.0 Kanamycin 100 27 485.3 → 163.1 Apramycin (IS) 109 14 540.3 → 378.2
41
The immunoassay analysis for amikacin was performed using a validated immunoassay method (Architect, Abbott Diagnostics, Chicago, IL, USA). This method was validated based on the FDA guidelines [16]. The LLOQ of this method was 1500 ng/mL. Accuracy and precision was determined on 4 concentration levels (11,500, 6,500, 15,000 and 27,500 ng/mL). The bias varied from -1.5% to 6.7% over all concentration levels. Within-run CV varied from 0.5% – 8.5% and between-run from 0.0 – 2.4%.
Analytical method validation
The method was validated based on to the Guidance for Industry Bioanalytical Method
Validation, published by the Food and Drug Administration (FDA).16 Since the FDA guidelines lack
requirements on the stability, a maximum bias of 15% was used according the EMA guidelines.17
The calibration line consisted of 8 concentration levels: 250-500-2,500-5,000-10,000-15,000-20,000-25,000 ng/mL for amikacin and 100-500-2,500-5,000-10,000-15,000-20,000-25,000 ng/mL for kanamycin. The upper limit was chosen as the linearity of the calibration line was not sufficient above 25,000 ng/mL. Stock standards and working solutions were diluted with serum to get the desired concentrations. Samples with concentrations above 25,000 ng/mL were diluted to obtain a concentration within the limits of the calibration line. Four QC samples were used at LLOQ (100 and 250 ng/mL for kanamycin and amikacin), LOW (500 ng/mL), MED (10,000 ng/mL) and HIGH (20,000 ng/mL). Calibration lines, correlation coefficients and regression coefficients were calculated using one-way ANOVA. Concentrations of the samples were calculated based on the peak height ratio with the internal standard with the compiled formula.
Selectivity was determined by analyzing six serum samples, each obtained from a different pool of serum. The extent of ion suppression or enhancement was tested, during a constant infusion of amikacin or kanamycin with IS, by injecting six pooled serum samples. Accuracy was evaluated by replicate analysis of the QC samples at all four concentration levels (LLOQ, LOW, MED and HIGH) on three consecutive days. The mean difference between the nominal concentration and the experimental result should be <15% (LLOQ <20%) [16]. Precision was determined using the measurements originating from the accuracy evaluation. The coefficient of variation (CV) was calculated at each individual concentration level. The CV should be lower than 15% (LLOQ < 20%).16
Recovery was determined by comparing the mean peak area of spiked blank serum with the peak area of the spiked extract serum. Recovery was determined at LOW, MED and HIGH levels of both amikacin and kanamycin. Matrix effect was assessed by dividing the peak area of spiked extract of blank serum by the peak area of spiked extraction fluid. Matrix effects were also determined at LOW, MED and HIGH concentration levels of amikacin and kanamycin. Both recovery and matrix effects were determined in five-fold.
Blank serum was spiked with amikacin or kanamycin at two concentration levels, LOW and HIGH. Stability was determined after three freeze-thaw cycles by measuring the spiked samples and assessing the difference with the nominal concentration. Short-term stability over 24 hours was tested at room temperature. The post-preparative stability was tested by analyzing spiked samples that had been placed in the autosampler for 24 hours. Concentrations were compared with calibration standards, which were freshly prepared on the day of the analysis. The mean of each stability test should be within 15% of the nominal concentration, according to EMA
guidelines.17 To assess the dilution integrity, a solution of 25,000 ng/mL amikacin and kanamycin
in serum was diluted in tenfold with serum to a 2,500 ng/mL solution and subsequently analyzed on three subsequent days.
42
Comparison between immunoassay and LC-MS/MS methods
To compare both methods, 17 clinical samples were measured with the new LC-MS/MS method and with the immunoassay. Only amikacin was compared, since the immunoassay is unable to analyze kanamycin.
The immunoassay analysis for amikacin was performed using a validated immunoassay method (Architect, Abbott Diagnostics, Chicago, IL, USA). This method was validated based on the FDA guidelines [16]. The LLOQ of this method was 1500 ng/mL. Accuracy and precision was determined on 4 concentration levels (11,500, 6,500, 15,000 and 27,500 ng/mL). The bias varied from -1.5% to 6.7% over all concentration levels. Within-run CV varied from 0.5% – 8.5% and between-run from 0.0 – 2.4%.
Clinical pilot
Routine TDM for TB drugs is performed for all patients with MDR-TB in the Tuberculosis Centre Beatrixoord, University Medical Center Groningen (Haren, the Netherlands). Routine TDM consists of sampling at 0, 1, 2, 3, 4 and 7 hours after administration. Medical charts were reviewed to detect patients that received amikacin or kanamycin as part of their TB treatment at our TB center and that were subjected to routine TDM. This study was evaluated by the local ethics committee (IRB 2013-492) and was according to the Dutch law allowed due to its retrospective nature.
Statistics
Validation parameters were calculated using a validated Excel sheet (Microsoft, Redmond, WA). Both the test for normality (Shapiro-Wilk test) as the The Wilcoxon Signed Rank Test were performed using SPSS 20 (SPSS, Virginia, IL).
RESULTS
Method validation
Three chromatograms of the LOW concentration (500 ng/mL) of amikacin, kanamycin and apramycin are displayed in figure 1. The retention times of amikacin, kanamycin and apramycin were determined: 2.53, 2.62 and 3.33 minutes. At these retention times, no other peaks are observed in pooled serum samples. The validated LLOQs were 250 ng/mL for amikacin and 100 ng/mL for kanamycin. Ion suppression or enhancement was tested using the constant infusion test.18 No ion suppression or enhancement was observed at the retention times of amikacin and kanamycin as displayed in figure 1.
Calibration of both amikacin and kanamycin was performed with eight concentration levels. Resulting calibration lines are displayed in table 3. Accuracy was determined by analyzing five different spiked samples at the four different QC concentration levels. Calculated biases were -8.8 – +7.0% and -6.7 – +5.6% for amikacin and kanamycin, respectively. All results are displayed in table 4. Precision was determined using data originating from the accuracy evaluation. The CV of the within-run and the between-run were calculated and are displayed in table 4. The CV varied between 1.7% – 6.7% for amikacin and 0.0% – 4.9% for kanamycin.
43
Figure 1. Three chromatograms of a LOW (500 ng/mL) concentration of amikacin, kanamycin and apramycin and the ion suppression test
Table 3. Calibration curves Compound Slope
(± st. dev) Intercept (± st. dev) Correlation coefficient Amikacin 0.761 ± 0.0168 -0.0545 ± 0.0105 0.995 Kanamycin 0.997 ± 0.0143 0.00390 ± 0.00391 0.997
44
The recovery of amikacin and kanamycin varied between 93.6 – 98.8%, as displayed in
table 4. Matrix effects were determined in five-fold and varied between 98.5 – 103.0%.
Stability of both analytes was assessed using the above proposed method. Measured concentrations differed from the nominal concentration with a maximal difference of -11.7 – +1.2% at room temperature, 1.3 – 7.4% in the autosampler and -2.6 – 10.8% after three freeze-thaw cycles. Dilution integrity was assessed on three different days in fivefold. The bias found was -8.0% and 5.0% for amikacin and kanamycin, respectively. Within-run CV was 2.6%, between-run CV varied from 1.7% to 2.9%.
Comparison between immunoassay (Architect) and LC-MS/MS
Results of both methods for quantification of amikacin are compared using the Wilcoxon Signed Rank Test, since the data was not normally distributed (P < 0.001, Shapiro-Wilk test). Test results from the immunoassay which were below the detection limit, were assumed to be 0. No significant difference was found (P = 0.501, n = 17). Therefore, no significant difference was found between the outcomes of both methods. However, the LLOQ of amikacin is on the LC-MS/MS lower than on the immunoassay.
Clinical pilot
Full pharmacokinetic concentrations curves were obtained of 2 patients using amikacin and 4
Table 4. Validation results of the LC-MS/MS method Criteria Concentration level
LLOQ LOW MED HIGH
Nominal concentration (ng/mL) Amikacin 250 500 10,000 20,000 Kanamycin 100 500 10,000 20,000 Accuracy (bias (%), n = 15) Amikacin 7.0 -8.8 2.6 -1.3 Kanamycin -3.9 -6.7 5.6 -0.9 Within-day precision (CV (%), n = 15) Amikacin 2.9 2.5 4.5 1.9 Kanamycin 4.9 2.9 4.0 2.1 Between-day precision (CV (%), n = 15) Amikacin 4.0 1.7 4.0 6.7 Kanamycin 0.0 3.5 1.8 2.8 Recovery (%) Amikacin n.a. 98.8 96.4 93.6 Kanamycin n.a. 98.4 97.1 93.9 Matrix effect (%) Amikacin n.a. 99,6 103.0 98,9 Kanamycin n.a. 99,2 98,7 98,5
Autosampler stability (24h) (bias %)
Amikacin n.a. 7.4 n.a. 5.5
Kanamycin n.a. 5.6 n.a. 1.3
Bench top stability (24h) (bias %)
Amikacin n.a. -11.7 n.a. 1.2
Kanamycin n.a. 1.1 n.a. -0.1
Freeze-thaw stability (after three freeze-thaw cycles) (bias %)
Amikacin n.a. -0.1 n.a. -2.6
Kanamycin n.a. 10.8 n.a. -2.1
n.a.: not available
N=15 means 5 replicate analyses on each day on each of the 3 validation days. Within-run and between-run coefficient of variation (CV) were calculated with One-Way ANOVA.
45
Figure 2. Concentration – time curve of MDR-TB patients receiving amikacin and kanamycin measured with LC-MS/MS.
patients using kanamycin, as shown in figure 2. This population consisted of five male patients and one female patient with a mean age of 33.7 (range 19 - 67). The mean BMI was 20.2 (range 13.0 – 26.5).
DISCUSSION
Method validation
This is the first LC-MS/MS method in literature to analyze amikacin and kanamycin in serum with minimal sample preprocessing. This LC-MS/MS method is validated on accuracy, precision, recovery, dilution integrity, matrix effect, autosampler stability, bench top stability and
46
freeze-thaw stability, based on the FDA guidelines.16 Concerning the stability, the FDA guidelines
do not contain exact limits. Therefore, the limits of the EMA guidelines were applied.17
The requirements which were demanded for this method complicated the development. In order to minimize the total processing time, SPE was avoided and a simple sample pre-processing method was developed. Furthermore, gradient elution was optimized to shorten the time of analysis. However, this method still has an adequate LLOQ for TDM and provides high sensitivity and selectivity.
The LLOQs are 250 ng/mL for amikacin and 100 ng/mL for kanamycin. These LLOQs are
comparable to a previous study (100 ng/mL)12, but significantly lower in comparison with the
method developed by Baietto et al. (2,340 ng/mL)13 and another recently published method (2.5
ng/mL).15 Furthermore, the LLOQ of amikacin was lower than the LLOQ of the immunoassay. The
LLOQs found in this study are sufficient to detect any clinically relevant trough levels. The ability to measure below 1500 ng/mL makes this method suitable for detecting trends in rising trough levels early and to adjust the dose accordingly.
Another advantage of this method is that other second line TB drugs, such as linezolid19,
clarithromycin20 and moxifloxacin21 can be monitored using LC-MS/MS. This in contrast to the
immunoassay, which provides only the quantification of amikacin. As described in the Global
Plan to Stop TB, laboratory strengthening is one of the core components.11 With this single
LC-MS/MS, TDM of these anti-TB drugs can be performed to monitor efficacy and possibly reduce toxicity. Further research could elucidate how to use dried blood spot to monitor aminoglycoside concentrations; simplifying storage and transport of blood samples, as already done for linezolid and moxifloxacin.22–25
The sample processing method used in this study is less time-consuming than the SPE
methods previously described.12 The recoveries found in this study were higher than in the study
of Baietto (85.2%), who used similar sample preprocessing.13 The method of Baietto required an
extra dilution step, which may introduce an additional error.13
During this study, apramycin was used as IS. Since apramycin is structurally related to
amikacin and kanamycin (figure 3a, b and c), it is our opinion that apramycin is suitable as IS.14
Apramycin is neither registered within the European Union nor in the United States for human use. A structural analogue as internal standard may not compensate as well for ion suppression effects as a deuterated internal standard. This may be because of the different retention time and ionization characteristics. However, the ion suppression tests showed that there was less than ±3% ion suppression, indicating that the developed method is free from ion suppression and that the used internal standard is suitable for its use.
Identical samples were measured with the immunoassay (Architect) and the new LC-MS/ MS method. A Wilcoxon Signed Rank test has been used to compare the two methods. With this statistical test, no significant difference has been found in the measured concentrations.
The newly validated LC-MS/MS enables the use of TDM with amikacin or kanamycin. Moreover, the new method is more sensitive in comparison with the immunoassay, providing information about trough levels.
CONCLUSION
This new method enables one to analyse amikacin and kanamycin both with one single method of analysis in a robust and reliable way. Moreover, the LLOQ of this method is sufficient to measure trough levels of both aminoglycosides.
47
Figure 3a. Chemical structure of apramycin
Figure 3c. Chemical structure of kanamycin
48
REFERENCES
1. Keshavjee S, Farmer PE. Tuberculosis, drug resistance, and the history of modern medicine. N Engl J Med 2012; 367(10): 931-6.
2. World Health Organization. Group of drugs to treat MDR-TB. In: Treatment of Tuberculosis guidelines. 4th ed. Switzerland: WHO Press; 2010. p.84.
3. Jureen P, Angeby K, Sturegard E, et al. Wild-type MIC distributions for aminoglycoside and cyclic polypeptide antibiotics used for treatment of Mycobacterium tuberculosis infections. J Clin Microbiol 2010; 48(5): 1853-8.
4. Craig WA. Does the dose matter? Clin Infect Dis 2001; 33 Suppl 3: S233-7.
5. Nuermberger E, Grosset J. Pharmacokinetic and pharmacodynamic issues in the treatment of mycobacterial infections. Eur J Clin Microbiol Infect Dis 2004; 23(4): 243-55.
6. Peloquin CA, Berning SE, Nitta AT, et al. Aminoglycoside toxicity: daily versus thrice-weekly dosing for treatment of mycobacterial diseases. Clin Infect Dis 2004; 38(11): 1538-44.
7. Perletti G, Vral A, Patrosso MC, et al. Prevention and modulation of aminoglycoside ototoxicity (Review).
Mol Med Report 2008; 1(1): 3-13.
8. de Jager P, van Altena R. Hearing loss and nephrotoxicity in long-term aminoglycoside treatment in patients with tuberculosis. Int J Tuberc Lung Dis 2002; 6(7): 622-7.
9. Gatell JM, Ferran F, Araujo V, Bonet M, Soriano E, Traserra J, SanMiguel JG. Univariate and multivariate analyses of risk factors predisposing to auditory toxicity in patients receiving aminoglycosides. Antimicrob
Agents Chemother 1987; 31(9): 1383-7.
10. Peloquin CA. Therapeutic drug monitoring in the treatment of tuberculosis. Drugs 2002; 62(15): 2169-83. 11. Stop TB Partnership, World Health Organization. The Global Plan to Stop TB 2011-2015. ; Available at: http://www.stoptb.org/assets/documents/global/plan/TB_GlobalPlanToStopTB2011-2015.pdf. Accessed 10/15, 2013.
12. Oertel R, Neumeister V, Kirch W. Hydrophilic interaction chromatography combined with tandem-mass spectrometry to determine six aminoglycosides in serum. J Chromatogr A 2004; 1058(1-2): 197-201.
13. Baietto L, D’Avolio A, De Rosa FG, et al. Development and validation of a simultaneous extraction procedure for HPLC-MS quantification of daptomycin, amikacin, gentamicin, and rifampicin in human plasma.
Anal Bioanal Chem 2010; 396(2): 791-8.
14. Lowes S, Jersey J, Shoup R, et al. Recommendations on: internal standard criteria, stability, incurred sample reanalysis and recent 483s by the Global CRO Council for Bioanalysis. Bioanalysis 2011; 3(12): 1323-32.
15. Han M, Jun SH, Lee JH, Park KU, Song J, Song SH. Method for simultaneous analysis of nine second-line anti-tuberculosis drugs using UPLC-MS/MS. J Antimicrob Chemother 2013; .
16. U.S. Departement of Health and Human Services, Food and Drug Administration. Guidance for industry, bioanalytical method validation. Rockville: Center for Drug Evaluation and Research; 2001.
17. European Medicines Agency, Committee for Medicinal Products for Human Use. Guideline on bioanalytical validation (EMEA/CHMP/EWP/192217/2009). London: European Medicines Agency; 2011. 18. Mallet CR, Lu Z, Mazzeo JR. A study of ion suppression effects in electrospray ionization from mobile phase additives and solid-phase extracts. Rapid Commun Mass Spectrom 2004; 18(1): 49-58.
49 19. Harmelink IM, Alffenaar JWC, Wessels AMA, Greijdanus B, Uges DRA. A rapid and simple liquid chromatography-tandem mass spectrometry method for the determination of linezolid in human serum. Eur
J Hosp Pharm 2008; 14: 5-8.
20. de Velde F, Alffenaar JW, Wessels AM, Greijdanus B, Uges DR. Simultaneous determination of clarithromycin, rifampicin and their main metabolites in human plasma by liquid chromatography-tandem mass spectrometry. J Chromatogr B Analyt Technol Biomed Life Sci 2009; 877(18-19): 1771-7.
21. Pranger AD, Alffenaar JW, Wessels AM, Greijdanus B, Uges DR. Determination of moxifloxacin in human plasma, plasma ultrafiltrate, and cerebrospinal fluid by a rapid and simple liquid chromatography- tandem mass spectrometry method. J Anal Toxicol 2010; 34(3): 135-41.
22. Alffenaar JW. Dried blood spot analysis combined with limited sampling models can advance therapeutic drug monitoring of tuberculosis drugs. J Infect Dis 2012; 205(11): 1765,6; author reply 1766.
23. Vu DH, Alffenaar JW, Edelbroek PM, Brouwers JR, Uges DR. Dried blood spots: a new tool for tuberculosis treatment optimization. Curr Pharm Des 2011; 17(27): 2931-9.
24. Vu DH, Bolhuis MS, Koster RA, et al. Dried blood spot analysis for therapeutic drug monitoring of linezolid in patients with multidrug-resistant tuberculosis. Antimicrob Agents Chemother 2012; 56(11): 5758-63. 25. Vu DH, Koster RA, Alffenaar JW, Brouwers JR, Uges DR. Determination of moxifloxacin in dried blood spots using LC-MS/MS and the impact of the hematocrit and blood volume. J Chromatogr B Analyt Technol
