Ultra-high throughput dual channel liquid chromatography with tandem mass spectrometry for quantification of four immunosuppressants in whole blood for therapeutic drug monitoring

Liquid chromatography with tandem mass spectrometry (LC-MS/MS) is the golden standard for immunosuppressants analyses, where optimising throughput by parallel chromatography can reduce costs and turnaround time. We aimed to double our system throughput using a dual LC-MS/MS setup. Therefore, two independent UPLC systems were hyphenated to one triple quadrupole MS, with staggered injections from one autosampler on alternating columns. The method simultaneously measured the analytes tacrolimus, sirolimus, everolimus, and cyclosporin A in whole blood using isotope dilution, with a run time of 1.5 min. Using the dual LC-MS/MS system, net run-to-run time improved from 2.3 to 0.98 min per injection, where throughput increased from 26 to 61 injections per hour. For Performance Qualification, 1101 clinical samples were measured on the dual LC-MS/MS system in addition to the standard system, during a period of one month, and the results were compared using Passing Bablok regression and Bland Altman analysis. There was excellent agreement for all four analytes, with regression slopes of 0.98-1.02x and intercepts of -0.11-0.88 µg/L. Minor bias was demonstrated between the systems with mean differences from -0.93 to 1.43%. In conclusion, the throughput was doubled and idle MS time was reduced with good agreement to the standard system. Currently, the method is applied for clinical routine with frequent peak intensities of &gt;180 injections per day.</p

A mobile microvolume UV/visible light spectrophotometer for the measurement of levofloxacin in saliva

INTRODUCTION: Therapeutic drug monitoring (TDM) for personalized dosing of fluoroquinolones has been recommended to optimize efficacy and reduce acquired drug resistance in the treatment of MDR TB. Therefore, the aim of this study was to develop a simple, low-cost, robust assay for TDM using mobile UV/visible light (UV/VIS) spectrophotometry to quantify levofloxacin in human saliva at the point of care for TB endemic settings. METHODS: All experiments were performed on a mobile UV/VIS spectrophotometer. The levofloxacin concentration was quantified by using the amplitude of the second-order spectrum between 300 and 400 nm of seven calibrators. The concentration of spiked samples was calculated from the spectrum amplitude using linear regression. The method was validated for selectivity, specificity, linearity, accuracy and precision. Drugs frequently co-administered were tested for interference. RESULTS: The calibration curve was linear over a range of 2.5-50.0 mg/L for levofloxacin, with a correlation coefficient of 0.997. Calculated accuracy ranged from -5.2% to 2.4%. Overall precision ranged from 2.1% to 16.1%. Application of the Savitsky-Golay method reduced the effect of interferents on the quantitation of levofloxacin. Although rifampicin and pyrazinamide showed analytical interference at the lower limit of quantitation of levofloxacin concentrations, this interference had no implication on decisions regarding the levofloxacin dose. CONCLUSIONS: A simple UV/VIS spectrophotometric method to quantify levofloxacin in saliva using a mobile nanophotometer has been validated. This method can be evaluated in programmatic settings to identify patients with low levofloxacin drug exposure to trigger personalized dose adjustment

Saliva-based linezolid monitoring on a mobile UV spectrophotometer

Background: In TB, therapeutic drug monitoring (TDM) is recommended for linezolid; however, implementation is challenging in endemic settings. Non-invasive saliva sampling using a mobile assay would increase the feasibility of TDM. Objectives: To validate a linezolid saliva assay using a mobile UV spectrophotometer. Methods: The saliva assay was developed using NanoPhotometer NP80 (R) and linezolid concentrations were quantified using second-order derivative spectroscopy. Sample preparation involved liquid-liquid extraction of saliva, using saturated sodium chloride and ethyl acetate at 1:1:3 (v/v/v). The assay was validated for accuracy, precision, selectivity, specificity, carry-over, matrix effect, stability and filters. Acceptance criteria were bias and coefficient of variation (CV) Results: Linezolid concentrations correlated with the amplitude between 250 and 270 nm on the second-order derivative spectra. The linezolid calibration curve was Linear over the range of 3.0 to 25 mg/L (R-2 = 0.99) and the LLOQ was 3.0 mg/L. Accuracy and precision were demonstrated with bias of -7.5% to 2.7% and CV Conclusions: We validated a UV spectrophotometric assay using non-invasive saliva sampling for linezolid. The next step is to demonstrate clinical feasibility and value to facilitate programmatic implementation of TDM

Development and validation of a simple LC-MS/MS method for simultaneous determination of moxifloxacin, levofloxacin, prothionamide, pyrazinamide and ethambutol in human plasma

Treatment of multidrug-resistant tuberculosis (MDR-TB) is challenging due to high treatment failure rate and adverse drug events. This study aimed to develop and validate a simple LC-MS/MS method for simultaneous measurement of five TB drugs in human plasma and to facilitate therapeutic drug monitoring (TDM) in MDR-TB treatment to increase efficacy and reduce toxicity. Moxifloxacin, levofloxacin, prothionamide, pyrazinamide and ethambutol were prepared in blank plasma from healthy volunteers and extracted using protein precipitation reagent containing trichloroacetic acid. Separation was achieved on an Atlantis T3 column with gradient of 0.1% formic acid in water and acetonitrile. Drug concentrations were determined by dynamic multiple reaction monitoring in positive ion mode on a LC-MS/MS system. The method was validated according to the United States' Food and Drug Administration (FDA) guideline for bioanalytical method validation. The calibration curves for moxifloxacin, levofloxacin, prothionamide, pyrazinamide and ethambutol were linear, with the correlation coefficient values above 0.993, over a range of 0.1-5, 0.4-40, 0.2-10, 2-100 and 0.2-10 mg/L, respectively. Validation showed the method to be accurate and precise with bias from 6.5% to 18.3% for lower limit of quantification and -5.8% to 14.6% for LOW, medium (MED) and HIGH drug levels, and with coefficient of variations within 11.4% for all levels. Regarding dilution integrity, the bias was within 7.2% and the coefficient of variation was within 14.9%. Matrix effect (95.7%-112.5%) and recovery (91.4%-109.7%) for all drugs could be well compensated by their isotope-labelled internal standards. A benchtop stability test showed that the degradation of prothionamide was over 15% after placement at room temperature for 72 h. Clinical samples (n = 224) from a cohort study were analyzed and all concentrations were within the analytical range. The signal of prothionamide was suppressed in samples with hemolysis which was solved by sample dilution. As the method is robust and sample preparation is simple, it can easily be implemented to facilitate TDM in programmatic MDR-TB treatment

Levofloxacin pharmacokinetics in saliva as measured by a mobile microvolume UV spectrophotometer among people treated for rifampicin-resistant TB in Tanzania

Background: Early detection and correction of low fluoroquinolone exposure may improve treatment of MDR-TB. Objectives: To explore a recently developed portable, battery-powered, UV spectrophotometer for measuring levofloxacin in saliva of people treated for MDR-TB. Methods: Patients treated with levofloxacin as part of a regimen for MDR-TB in Northern Tanzania had serum and saliva collected concurrently at 1 and 4 h after 2 weeks of observed levofloxacin administration. Saliva levofloxacin concentrations were quantified in the field via spectrophotometry, while serum was analysed at a regional laboratory using HPLC. A Bayesian population pharmacokinetics model was used to estimate the area under the concentration-time curve (AUC(0-24)). Subtarget exposures of levofloxacin were defined by serum AUC(0-24) Results: Among 45 patients, 11 (25.6%) were women and 16 (37.2%) were living with HIV. Median AUC(0- 24) in serum was 140 (IQR = 102.4-179.09) mg.h/L and median AUC(0- 24) in saliva was 97.10 (IQR = 74.80-121.10) mg.h/L. A positive linear correlation was observed with serum and saliva AUC(0-24), and a receiver operating characteristic curve constructed to detect serum AUC(0- 24) below 80mg.h/L demonstrated excellent prediction [AUC 0.80 (95% CI = 0.62-0.94)]. Utilizing a saliva AUC(0- 24) cut-off of 91.6mg.h/L, the assay was 88.9% sensitive and 69.4% specific in detecting subtarget serum AUC(0- 24) values, including identifying eight of nine patients below target. Conclusions: Portable UV spectrophotometry as a point-of-care screen for subtarget levofloxacin exposure was feasible. Use for triage to other investigation or personalized dosing strategy should be tested in a randomized study

Corrigendum to "Mass spectrometry for therapeutic drug monitoring of anti-tuberculosis drugs" [Clin. Mass Spectrom. 14(Part A) (2019) 34-45]

[This corrects the article DOI: 10.1016/j.clinms.2018.10.002.]

Evaluation of saliva as a potential alternative sampling matrix for therapeutic drug monitoring of levofloxacin in MDR-TB patients

Saliva may be a useful alternative matrix for monitoring levofloxacin concentrations in multi-drug resistant TB patients. The objectives of this study were: a) to evaluate the correlation between plasma and salivary Lfx concentrations in MDR-TB patients; and b) to gauge the possibility of using saliva as an alternative sampling matrix for therapeutic drug monitoring of Lfx in TB endemic areas. This was a prospective pharmacokinetic study that enrolled MDR-TB patients receiving levofloxacin (Lfx; 750-1000mg once daily dosing) under standardized treatment regimen in Nepal. Paired blood and saliva samples were collected at steady state. Lfx concentrations were quantified using liquid chromatography- tandem mass spectrometry. Pharmacokinetic parameters were calculated using non-compartmental kinetics. Lfx drug exposure was evaluated in 23 MDR-TB patients. During the first month, the median (IQR) area under the concentration-time curve (AUC0-24) was 67.09 (53.93-98.37) mg*h/L in saliva and 99.91 (76.80-129.70) mg*h/L in plasma, and the saliva plasma (S/P) ratio was 0.69 (0.53-0.99). Similarly, during the second month, the median (IQR) AUC0-24 was 75.63 (61.45-125.5) mg*h/L in saliva and 102.7 (84.46-131.9) mg*h/L in plasma with a S/P ratio of 0.73 (0.66-1.18). Furthermore, large inter-and intra-individual variabilities in Lfx concentrations were observed. This study could not demonstrate a strong correlation between plasma and saliva Lfx levels. Despite a good Lfx penetration in saliva, the variability in individual saliva-to-plasma ratios limits the use of saliva as a valid substitute for plasma. Nevertheless, saliva could be useful in semi-quantitatively predicting Lfx plasma levels

Quality Assessment of Dried Blood Spots from Tuberculosis Patients from Four Countries

BACKGROUND: Dried blood spot (DBS) sampling is a blood collection tool that uses a finger prick to obtain a blood drop on a DBS card. It can be used for therapeutic drug monitoring, a method that uses blood drug concentrations to optimize individual treatment. DBS sampling is believed to be a simpler way of blood collection compared with venous sampling. The aim of this study was to evaluate the quality of DBSs from patients with tuberculosis all around the world based on quality indicators in a structured assessment procedure. METHODS: Total 464 DBS cards were obtained from 4 countries: Bangladesh, Belarus, Indonesia, and Paraguay. The quality of the DBS cards was assessed using a checklist consisting of 19 questions divided into 4 categories: the integrity of the DBS materials, appropriate drying time, blood volume, and blood spot collection. RESULTS: After examination, 859 of 1856 (46%) blood spots did not comply with present quality criteria. In 625 cases (34%), this was due to incorrect blood spot collection. The DBS cards from Bangladesh, Indonesia, and Paraguay seemed to be affected by air humidity, causing the blood spots not to dry appropriately. CONCLUSIONS: New tools to help obtain blood spots of sufficient quality are necessary and environmental specific recommendations to determine plasma concentration correctly. In addition, 3% of the DBS cards were rejected because the integrity of the materials suggesting that the quality of plastic ziplock bags currently used to protect the DBS cards against contamination and humidity may not be sufficient