Development and validation of a fast and sensitive UHPLC-DAD assay for the quantification of nitrofurantoin in plasma and urine.

Nitrofurantoin is an antimicrobial drug that has been used in the treatment of lower urinary tract infections for more than 50 years. Despite its long use, surprisingly little is known of the pharmacokinetics of nitrofurantoin, whereas this is essential to optimize patient treatment. We developed a novel analytical method for the quantification of nitrofurantoin in plasma and urine using ultra-high performance liquid chromatography and diode array detection to allow pharmacokinetic studies in these two matrices. The sample preparation method consisted of protein precipitation for plasma and liquid-liquid extraction for urine. 100 µL was needed for the sample preparation. Furazolidone was used as internal standard. Gradient chromatographic separation was performed on a HSS-T3 column. UV detection was performed at a wavelength of 369 nm. The analysis time was 5 min. The method was successfully validated according to the FDA-guidelines (2018). Linearity was confirmed over a concentration range from 50 to 1250 µg/L in plasma and from 4 to 200 mg/L in urine (r2 > 0.95). Validation results of five QC concentrations for plasma and urine, respectively, are for within-day accuracy <± 13% in both matrices, for between-day accuracy <± 7% and <± 9%, for within-day precision <10% and <4% and for between-day precision <10% and <5%. Plasma samples are stable for seven days at 4 °C, and for 2 years at -20 °C and-80 °C. Urine samples are stable for at least seven days at 4 °C and at room temperature and for 2 years at -20 °C andat -80 °C, except from the lower concentrated samples, which are only stable at -80 °C. All samples were kept from daylight using amber colored glassware. The presented method meets all validation requirements and was successfully used in a clinical study where the pharmacokinetics of nitrofurantoin were investigated in healthy volunteers. The easy sample preparation method and the short analysis time make this method suitable for use during routine clinical practice to study the pharmacokinetics of nitrofurantoin.

Dried Blood Spot Sampling for Tacrolimus and Mycophenolic Acid in Children: Analytical and Clinical Validation.

BACKGROUND: Tacrolimus and mycophenolic acid (MPA) are the backbone of immunosuppressive therapy after pediatric kidney transplantation. Dosing of these drugs is individualized by therapeutic drug monitoring. Dried blood spot (DBS) sampling may prove beneficial over conventional venous sampling. We aimed to develop and clinically validate a DBS method for tacrolimus and MPA in children. METHODS: A joint DBS liquid chromatography-mass spectrometry assay for tacrolimus and MPA was developed. DBS-specific items included the hematocrit effect and influence of spot volume. Subsequently, a clinical validation study among children aged 2-18 years was performed to assess the agreement between observed and DBS-predicted venous concentrations. Agreement of the methods was assessed with Passing-Bablok regression, Bland-Altman plots, and quantification of the DBS predictive performance in terms of bias (median percentage prediction error) and precision (median absolute percentage prediction error), both should be <15%. RESULTS: A total of 40 tacrolimus and 32 MPA samples were available from 28 children. Conversion factors were used to predict venous concentrations from DBS. For tacrolimus, 95% of the individual ratios of predicted and observed concentrations were within a range of 0.74-1.28, with 85% of these ratios between 0.80 and 1.20 (Bland-Altman plots). For MPA, the 95% limits of agreement represented a broader range of 0.49-1.49%, and 72% of individual ratios were between the 0.80 and 1.20 limits. Median percentage prediction error and median absolute percentage prediction error were less than 15% for both drugs. CONCLUSIONS: A DBS assay was developed for tacrolimus and MPA. Tacrolimus venous concentrations could be adequately predicted from DBS. DBS analysis of MPA seemed to be a semiquantitative measurement at the most when compared with conventional plasma analysis, considering the high variability between observed and predicted concentrations. Next, home-based DBS sampling of tacrolimus for the purpose of therapeutic drug monitoring will be implemented into routine clinical care.

Pharmacokinetic drug-drug interaction study between raltegravir and citalopram.

BACKGROUND: Depression is the most common mental health disorder among HIV-infected patients. When treating HIV-infected patients with a selective serotonin reuptake inhibitor (SSRI), potential drug-drug interactions with antiretroviral agents have to be taken into account. We investigated the two-way pharmacokinetic drug-drug interaction and tolerability of concomitant administration of the SSRI citalopram and the HIV-1 integrase inhibitor raltegravir in healthy volunteers. METHODS: An open-label, crossover, two-period trial was conducted in 24 healthy volunteers. Subjects received the following treatments: citalopram 20 mg once daily for 2 weeks followed by the combination with raltegravir 400 mg twice daily for 5 days and after a washout period raltegravir 400 mg twice daily for 5 days. Intensive steady-state pharmacokinetic blood sampling was performed. Geometric mean ratios (GMRs) of the combination versus the reference treatment and 90% CIs were calculated for the area under the plasma concentration-time curve (AUC). CYP2C19 genotyping was performed because it influences N-demethylation of citalopram to desmethylcitalopram. RESULTS: A total of 22 healthy volunteers completed the trial. GMRs (90% CI) were 1.00 (0.98, 1.03) for citalopram AUC0-24 h, 0.99 (0.88, 1.12) for desmethylcitalopram AUC0-24 h and 0.77 (0.50, 1.19) for raltegravir AUC0-12 h. Raltegravir plasma concentration 12 h after intake (C12 h) did not change with concomitant use of citalopram. Within each CYP2C19 phenotype subgroup the citalopram metabolite-to-parent ratio, which is a measure for metabolic enzyme activity, was not influenced by concomitant raltegravir use. CONCLUSIONS: Raltegravir does not influence the pharmacokinetics of citalopram and desmethylcitalopram. Citalopram did not change the pharmacokinetics of raltegravir in a clinically meaningful way. The combination was well tolerated and can be administered without dose adjustments. ClinicalTrials.gov NCT01978782.

Delayed trough level measurement with the use of prolonged-release tacrolimus.

Trough concentrations of prolonged-release tacrolimus are usually measured at 24 h after taking the drug in the morning. It is impractical to measure these trough concentrations in patients who visit the outpatient clinic in the afternoon. Trough concentrations obtained in the afternoon may also be suitable for estimating the 24-h exposure. We therefore aimed to assess the usefulness of tacrolimus concentrations measured at 32 h postdose for therapeutic drug monitoring in renal transplant patients who take prolonged-release tacrolimus. We measured tacrolimus pharmacokinetics in 26 patients using prolonged-release tacrolimus. Eleven blood samples were taken during a period of 32 h after ingestion by use of a validated dried blood spot method. Tacrolimus concentrations were measured with HPLC-tandem mass spectrometry. The mean concentrations at 24 and 32 h postdose were 8.3 µg/l (7.5-9.1) and 6.7 µg/l (6.1-7.4), respectively (P < 0.0001). The Spearman correlation coefficients between these concentrations and 24-h exposure were 0.83 and 0.82, respectively (both P < 0.01). In conclusion, delayed trough level measurement provides lower values and therefore requires adjustment of the target range. However, levels measured until 32 h after ingestion remain strongly correlated with 24-h exposure. This warrants the use of delayed trough level measurement to improve patient convenience.

Effect of mild diarrhea on tacrolimus exposure.

BACKGROUND: Diarrhea is a frequent adverse event in patients treated with the combination of tacrolimus and mycophenolate mofetil (MMF). In case of severe diarrhea, the total exposure to tacrolimus can substantially increase, which is reflected in a rise of the predose trough level (C0). In mild diarrhea (two to three stools per day), an increased exposure might occur without trough levels exceeding the target range, resulting in "silent" chronic tacrolimus overexposure. The aim was to assess the degree of unnoticed tacrolimus overexposure in renal transplant patients with mild diarrhea while on treatment with tacrolimus and MMF. METHODS: A prospective pharmacokinetic study was performed in 12 recipients of a renal allograft using a combination of tacrolimus and MMF with mild diarrhea and in 12 controls. Tacrolimus levels were assessed by a validated dried blood spot method for sampling and measurement. RESULTS: The C0 did not differ between patients with mild diarrhea and controls (mean [95% confidence interval], 9.6 µg/L [8.6-10.9 µg/L] and 8.3 µg/L [6.9-9.9 µg/L]). In addition, there was no significant difference in the 12-hr area under the curve between patients with mild diarrhea and controls (185.6 µg· h/L [153.6-224.2 µg·h/L] vs. 170.5 µg·h/L [137.2-221.8 µg·h/L]). As a result, the ratio between the 12-hr area under the curve and C0 was similar in both groups (19.2 [17.5-21.1] vs. 20.6 [19.0-22.4]). The intraindividual variability in tacrolimus exposure was limited and not affected by the presence of mild diarrhea. CONCLUSIONS: We found no evidence for the presence of hidden tacrolimus overexposure in patients with mild diarrhea while on treatment with tacrolimus and MMF.

Dried blood spot measurement: application in tacrolimus monitoring using limited sampling strategy and abbreviated AUC estimation.

Dried blood spot (DBS) sampling and high-performance liquid chromatography tandem-mass spectrometry have been developed in monitoring tacrolimus levels. Our center favors the use of limited sampling strategy and abbreviated formula to estimate the area under concentration-time curve (AUC(0-12)). However, it is inconvenient for patients because they have to wait in the center for blood sampling. We investigated the application of DBS method in tacrolimus level monitoring using limited sampling strategy and abbreviated AUC estimation approach. Duplicate venous samples were obtained at each time point (C(0), C(2), and C(4)). To determine the stability of blood samples, one venous sample was sent to our laboratory immediately. The other duplicate venous samples, together with simultaneous fingerprick blood samples, were sent to the University of Maastricht in the Netherlands. Thirty six patients were recruited and 108 sets of blood samples were collected. There was a highly significant relationship between AUC(0-12), estimated from venous blood samples, and fingerprick blood samples (r(2) = 0.96, P < 0.0001). Moreover, there was an excellent correlation between whole blood venous tacrolimus levels in the two centers (r(2) = 0.97; P < 0.0001). The blood samples were stable after long-distance transport. DBS sampling can be used in centers using limited sampling and abbreviated AUC(0-12) strategy as drug monitoring.

Genotyping with a dried blood spot method: a useful technique for application in pharmacogenetics.

Several commercial DNA isolation kits are available for extracting the genomic DNA from the ethylene diamine tetra-acetic acid (EDTA) whole blood samples. To obtain DNA from whole blood these DNA isolation procedures require quite some hands on time and are rather expensive. An alternative technique could be dried blood spot (DBS) sampling, with which DNA isolation is faster, cheaper and logistics are easier. We have developed a non-commercial DBS method and examined its performance in practice. DNA isolation from EDTA blood samples and made blood spots on filter paper from 106 renal transplant recipients were compared. Additionally, DNA isolation with a column method and two different DBS method was performed for 10 healthy volunteers and compared. Also DNA isolation with only capillary blood using both DBS methods from another 100 healthy volunteers has been investigated. Real-time PCR FRET assays for the CYP3A4 A-392G, CYP3A5 A6986G, ABCB1 C1236T, G2677T/A and C3435T polymorphisms were used and the melting curves of both DNA isolation methods were compared. In all cases DNA extracted with the column method corresponded completely with the results of the DNA isolated with the DBS procedure. Hence, DNA isolation from filter paper appears to be a useful alternative for the commercially available DNA isolation kits.

Dried blood spot measurement of tacrolimus is promising for patient monitoring.

The usefulness of dried blood spot (DBS) sampling for therapeutic drug monitoring of tacrolimus was investigated with renal transplant patients. There was no significant difference between the concentrations (ranging 3.33-53.9 mug/l) of 34 samples of 26 stable renal transplant outpatients, measured both after venous and DBS sampling. DBS sampling is easy to perform because concentrations with and without nurse assistance did not significantly differ. No significant difference was found between tacrolimus concentrations in 20 duplicate DBS samples before and after postal transport. DBS seems promising for routine patient monitoring.