Higher Tissue Concentrations of Vancomycin Achieved With Low-Dose Intraosseous Injection Versus Intravenous Despite Limited Tourniquet Duration in Primary Total Knee Arthroplasty: A Randomized Trial.

BACKGROUND: Vancomycin use has been suggested in high risk patients undergoing total knee arthroplasty (TKA). Previous literature has shown that a lower dose (500 mg) of vancomycin given by intraosseous regional administration (IORA) achieves tissue concentrations 4-10 times higher than intravenous (IV) administration. There is increasing interest in performing TKA with limited tourniquet inflation time. The purpose of this study is to evaluate whether IORA of vancomycin can achieve effective tissue concentrations with limited tourniquet inflation time. METHODS: Based on prior power calculations, 24 patients undergoing primary TKA were randomized into 2 groups. Group IV-Systemic received weight-based (15 mg/kg) vancomycin with the tourniquet inflated for cementation only. Group IORA received 500 mg vancomycin via IORA after tourniquet inflation which remained inflated for 10 minutes, then reinflated for cementation only. Vancomycin concentrations from tissue, serum, and drain fluid were compared between the 2 groups. RESULTS: Median vancomycin concentrations in tissue were significantly higher (5-15 times) at all time points in the IORA group. Concentrations in fat at the time of wound closure, after the tourniquet had been deflated for most of the procedure, were 5.2 µg/g in Group IV-Systemic and 33.1 µg/g in Group IORA (P < .001). Median bone concentrations taken just prior to cementation were 7.9 µg/g in Group IV-Systemic and 21.8 µg/g in Group IORA (P = .006). There were no complications related to IORA. CONCLUSION: For surgeons who wish to limit tourniquet time and when indicated to use vancomycin, low-dose vancomycin IORA achieves tissue concentrations 5-15 times higher than those achieved by IV administration. LEVEL OF EVIDENCE: Level 1 therapeutic randomized trial.

Simultaneous Determination of Dabigatran, Rivaroxaban, and Apixaban in Human Plasma by Liquid Chromatography/Tandem Mass Spectrometry.

BACKGROUND: Pharmacokinetic studies and therapeutic drug monitoring of anticoagulants require a simple, rapid, and reliable analytical method for monitoring plasma concentrations. The aims of the current work were to develop and validate a liquid chromatography/tandem mass spectrometry method for the simultaneous determination of 3 direct oral anticoagulants (dabigatran, rivaroxaban, and apixaban) in human plasma that is suitable for pharmacokinetic studies and routine therapeutic drug monitoring in busy hospital laboratories. METHODS: This method included a hydrolysis step to account for the active acylglucuronide metabolites of dabigatran that demonstrate an equivalent anticoagulant effect as dabigatran. After hydrolysis, a simple one-step protein precipitation was used for sample preparation. Total dabigatran (the sum of free dabigatran and the contribution from dabigatran acylglucuronides), rivaroxaban, and apixaban, and their corresponding isotopically labeled internal standards were resolved on a C18(2) column. All compounds were detected using electrospray ionization liquid chromatography/tandem mass spectrometry in the positive mode. RESULTS: For all 3 anticoagulants, standard curves were linear over the concentration range of 1.0-1000 mcg/L (r > 0.99), bias was < ±10%, and intraday and interday coefficients of variation (imprecision) were <10%. The limit of quantification was 1.0 mcg/L. For all 3 anticoagulants and corresponding isotopically labeled internal standards, the absolute recoveries were similar and consistent, with mean values of 93%-102%. No significant matrix effects were observed. CONCLUSIONS: This method is simple, rapid, robust, and reliable and can be used to analyze the plasma concentrations of the drugs in patients on dabigatran or rivaroxaban therapy.

Simultaneous Determination of Cefalexin, Cefazolin, Flucloxacillin, and Probenecid by Liquid Chromatography-Tandem Mass Spectrometry for Total and Unbound Concentrations in Human Plasma.

BACKGROUND: Pharmacokinetic studies and therapeutic drug monitoring of antibiotics require a simple, rapid, and reliable analytical method for monitoring the concentrations in plasma, including unbound concentrations for highly protein-bound drugs. The aim of the current work was to develop and validate a liquid chromatography-tandem mass spectrometry method for the simultaneous determination of total and unbound concentrations of 3 widely used ß-lactam antibiotics (cefalexin, cefazolin, and flucloxacillin) and the often coadministered drug probenecid in human plasma, suitable for pharmacokinetic studies and for routine use in ordinary, busy hospital laboratories. METHODS: Unbound drug was separated from bound drug by ultrafiltration. A simple 1-step protein precipitation was used for sample preparation. Cefalexin, cefazolin, flucloxacillin, probenecid, and their corresponding isotopically labeled internal standards were then resolved on a C18 (2) column. All the compounds were detected using electrospray ionization in the positive mode. RESULTS: Standard curves were linear for all compounds over the concentration range of 0.2-100 mg/L (r > 0.99) for total drug in plasma and 0.01-10 mg/L (r > 0.99) for unbound drug in plasma ultrafiltrate. For both total and unbound drugs, bias was <±10%, and intra- and interday coefficients of variation (imprecision) were <10%. The limit of quantification was 0.2 mg/L for total plasma concentrations and 0.01 mg/L for plasma ultrafiltrate concentrations of all drugs. CONCLUSIONS: The method has proven to be simple, rapid, robust, and reliable and is currently being used in clinical pharmacokinetic studies and in the routine clinical service to enhance the effective use of the ß-lactam antibiotics.

The AAHKS Clinical Research Award: Intraosseous Regional Prophylaxis Provides Higher Tissue Concentrations in High BMI Patients in Total Knee Arthroplasty: A Randomized Trial.

BACKGROUND: Obesity is an established risk factor for periprosthetic joint infections after total knee arthroplasty (TKA). In obese patients, a larger dose of prophylactic vancomycin based on actual body weight is required to reach therapeutic concentrations. It is unclear how tissue concentrations are affected when intraosseous regional administration (IORA) is used in this population. This study compared tissue concentrations of low-dose vancomycin via IORA vs actual body weight-adjusted systemic intravenous (IV) dose in primary TKA. METHODS: Twenty-two patients with a body mass index (BMI) >35 undergoing TKA were randomized into 2 groups. The IV group received 15 mg/kg (maximum of 2 g) of systemic IV vancomycin and the IORA group received 500 mg vancomycin into the tibia. Subcutaneous fat and bone samples were taken at regular intervals. Tissue antibiotic concentrations were measured using liquid chromatography coupled with tandem mass spectrometry. A blood sample was taken 1 to 2 hours after tourniquet deflation to measure systemic concentration. RESULTS: The mean BMI was 41.1 in the IORA group and 40.1 in the IV systemic group. The overall mean tissue concentration in subcutaneous fat was 39.3 µg/g in the IORA group and 4.4 µg/g in the IV systemic group (P < .01). Mean tissue concentrations in bones were 34.4 µg/g in the IORA group and 6.1 µg/g in the IV systemic group (P < .01). CONCLUSION: Low-dose IORA was effective in the high-BMI population group, providing tissue concentrations of vancomycin 5-9 times higher than systemic administration. IORA optimizes timing of vancomycin administration and provides high tissue antibiotic concentrations during TKA in this high-risk patient group.

The John N. Insall Award: Higher Tissue Concentrations of Vancomycin Achieved With Intraosseous Regional Prophylaxis in Revision TKA: A Randomized Controlled Trial.

BACKGROUND: In primary TKA, prophylaxis with low-dose vancomycin through intraosseous regional administration (IORA) achieves tissue concentrations six to 10 times higher than systemic administration and was shown to provide more effective prophylaxis in an animal model. However, in revision TKA, the presence of a tibial implant may compromise IORA injection, and tourniquet deflation during a prolonged procedure may lower tissue concentrations. QUESTIONS/PURPOSES: (1) Does low-dose IORA reliably provide equal or higher tissue concentrations of vancomycin compared with systemic IV administration in revision TKA? (2) Are tissue concentrations of vancomycin after IORA maintained for the duration of the revision TKA despite a period of tourniquet deflation? (3) Is there any difference in early postoperative (< 6 weeks) complications between IORA and systemic IV administration in revision TKA? METHODS: Twenty patients undergoing aseptic revision TKA were randomized to two groups. The IV group received 1 g systemic IV prophylactic vancomycin. The IORA group received 500 mg vancomycin as a bolus injection into a tibial intraosseous cannula below an inflated thigh tourniquet before skin incision. In all patients receiving IORA, intraosseous tibial injection was technically possible despite the presence of a tibial implant. Mean procedure length was 3.5 hours in both groups. Mean initial tourniquet inflation was 1.5 hours with a second inflation for a mean of 35 minutes during cementation. During the procedure, subcutaneous fat and bone samples were taken at regular intervals. Tissue vancomycin concentrations were measured using high-performance liquid chromatography. RESULTS: Overall geometric mean tissue concentration of vancomycin in fat samples was 3.7 µg/g (95% confidence interval [CI], 2.6-5.2) in the IV group versus 49.3 µg/g in the IORA group (95% CI, 33.2-73.4; ratio between means 13.5; 95% CI, 8.2-22.0; p < 0.001); mean tissue concentrations in femoral bone were 6.4 µg/g (95% CI, 4.5-9.2) in the IV group versus 77.1 µg/g (95% CI, 42.4-140) in the IORA group (ratio between means 12.0; 95% CI, 6.2-23.2; p < 0.001). Vancomycin concentrations in the final subcutaneous fat sample taken before closure were 5.3 times higher in the IORA group versus the IV group (mean ± SD, 18.2 ± 11.6 µg/g IORA versus 3.6 ± 2.5 µg/g; p < 0.001). The intraarticular concentration of vancomycin on postoperative Day 1 drain samples was not different between the two groups with the numbers available (mean 4.6 µg/L in the IV group versus 6.6 µg/g in the IORA group; mean difference 2.0 µg/g; 95% CI, 6.2-23.2; p = 0.08). CONCLUSIONS: IORA administration of vancomycin in patients undergoing revision TKA resulted in tissue concentrations of vancomycin five to 20 times higher than systemic IV administration despite the lower dose. High tissue concentrations were maintained throughout the procedure despite a period of tourniquet deflation. These preliminary results justify prospective cohort studies, which might focus on broader safety endpoints in more diverse patient populations. We believe that these studies should evaluate patients undergoing revision TKA in particular, because the risk of infection is greater than in patients undergoing primary TKA. LEVEL OF EVIDENCE: Level I, therapeutic study.

The Mark Coventry Award: Higher tissue concentrations of vancomycin with low-dose intraosseous regional versus systemic prophylaxis in TKA: a randomized trial.

BACKGROUND: In response to increasing antibiotic resistance, vancomycin has been proposed as an alternative prophylactic agent in TKA. However, vancomycin requires a prolonged administration time, risks promoting further antibiotic resistance, and can cause systemic toxicity. Intraosseous regional administration (IORA) is known to achieve markedly higher antibiotic concentrations than systemic administration and may allow the use of a lower vancomycin dose. QUESTIONS/PURPOSES: We assessed whether low-dose IORA vancomycin can achieve tissue concentrations equal or superior to those of systemic administration in TKA and compared complications between patients treated with IORA and intravenous vancomycin. METHODS: We randomized 30 patients undergoing primary TKA to receive 250 or 500 mg vancomycin via IORA or 1 g via systemic administration. IORA was performed as a bolus injection into a tibial intraosseous cannula below an inflated thigh tourniquet immediately before skin incision. Subcutaneous fat and bone samples were taken during the procedure and antibiotic concentrations measured. RESULTS: The overall mean tissue concentration of vancomycin in subcutaneous fat was 14 µg/g in the 250-mg IORA group, 44 µg/g in the 500-mg IORA group, and 3.2 µg/g in the systemic group. Mean concentrations in bone were 16 µg/g in the 250-mg IORA group, 38 µg/g in the 500-mg IORA group, and 4.0 µg/g in the systemic group. One patient in the systemic group developed red man syndrome during infusion. CONCLUSIONS: Low-dose IORA vancomycin results in tissue concentrations equal or superior to those of systemic administration. IORA optimizes timing of vancomycin administration, and the lower dose may reduce the risk of systemic side effects while providing equal or enhanced prophylaxis in TKA.

Thirteen years' experience of pharmacokinetic monitoring and dosing of busulfan: can the strategy be improved?

BACKGROUND: A busulfan concentration monitoring and dosing service has been provided by Christchurch Hospital since 1998. This study aimed to see (1) the percentage of patients with an area under the concentration time curve (AUC) outside the target range and had dose adjustment, (2) how busulfan clearance (CL) relates to body weight, and (3) if fewer samples could be used to predict doses. METHODS: Blood samples were taken from patients after oral administration, usually at 0.5, 1, 1.5, and 6 hours, and after the start of a 2-hour intravenous (IV) infusion of busulfan, at 1, 2, 2.5, 3, 6, and 8 hours. Dose adjustment was made based on the AUC compared with the target range. The relationship of CL and body weight for the IV group was used to develop a revised IV dosing schedule. The bias and imprecision of AUCs estimated using fewer sampling points were examined to see if sampling could be economized. RESULTS: Data were available for 150 patients but for 6 patients, data were incomplete and excluded. Of the remaining 144 patients (256 sample sets, 209 oral, 47 IV, 62% with repeats), 38% (IV) and 35% (oral) of patients had AUCs within the target range after the first dose. Dose adjustment was made in 47% and 34% of patients dosed IV and orally, respectively, after which there was a trend to more patients achieving the target AUC. A nonlinear relationship was found between CL and body weight. The initial IV dosing schedule was revised to take this into account. Sampling for busulfan concentration measurement at 3 points (2.5, 4, 8 hours) or 2 points (2.5, 8 hours) after the start of the infusion enabled accurate and precise estimates of AUC0₋24. CONCLUSIONS: Around two thirds of patients treated with busulfan were outside the target AUC range after the first dose. Dose adjustment was made in 37% of patients. The relationship between CL and body weight was used to revise the initial IV dosing schedule. Sampling for AUC estimation could be reduced to 2 time points after IV dosing.

A simple high-performance liquid chromatography method for simultaneous determination of three triazole antifungals in human plasma.

A rapid and simple high-performance liquid chromatography (HPLC) assay was developed for the simultaneous determination of three triazole antifungals (voriconazole, posaconazole, and itraconazole and the metabolite of itraconazole, hydroxyitraconazole) in human plasma. Sample preparation involved a simple one-step protein precipitation with 1.0 M perchloric acid and methanol. After centrifugation, the supernatant was injected directly into the HPLC system. Voriconazole, posaconazole, itraconazole, its metabolite hydroxyitraconazole, and the internal standard naproxen were resolved on a C(6)-phenyl column using gradient elution of 0.01 M phosphate buffer, pH 3.5, and acetonitrile and detected with UV detection at 262 nm. Standard curves were linear over the concentration range of 0.05 to 10 mg/liter (r(2) > 0.99). Bias was <8.0% from 0.05 to 10 mg/liter, intra- and interday coefficients of variation (imprecision) were <10%, and the limit of quantification was 0.05 mg/liter.

Determination of imatinib and its active metabolite N-desmethyl imatinib in human plasma by liquid chromatography/tandem mass spectrometry.

Imatinib is a first-line treatment for chronic myelogenous leukaemia (CML). The pharmacokinetics of imatinib in patients with CML are characterised by large interpatient variability. Concentration monitoring of imatinib and its active metabolite N-desmethyl imatinib (DMI) is considered necessary to enhance the safe and effective use of imatinib. A rapid, simple and sensitive liquid chromatography/tandem mass spectrometry assay was developed for the simultaneous determination of imatinib and its metabolite DMI in human plasma. After proteins were precipitated with acetonitrile, imatinib, DMI and the internal standard D8-imatinib were resolved on a Gemini-NX 3 µm C18 column using gradient elution of 0.05 % formic acid and methanol. The three compounds were detected using electrospray ionisation in the positive mode. Standard curves of imatinib and DMI were adequately fitted by quadratic equations (r > 0.999) over the concentration range of 10 to 2,000 ng/mL which encompasses clinical concentrations. Bias was ≤±8.3 %, intra- and inter-day coefficients of variation (imprecision) were ≤8.0 % and the limit of quantification was 10 ng/mL for both imatinib and DMI. The assay is being used successfully in clinical practice to enhance the safe and effective use of imatinib.

Determination of methylphenidate and its metabolite ritalinic acid in urine by liquid chromatography/tandem mass spectrometry.

Methylphenidate (MPH) is a drug that is licensed for treatment of ADHD and also narcolepsy. Monitoring of the parent drug and its major metabolite ritalinic acid (RA) in urine is considered necessary to ensure compliance with treatment programmes. A rapid, simple and sensitive liquid chromatography/tandem mass spectrometry (LC-MS/MS) assay was developed for the determination of MPH and its metabolite RA in human urine. After urine was diluted with water, methylphenidate, the major metabolite ritalinic acid, and d6-amphetamine as the internal standard were resolved on a PFP propyl column using gradient elution of 0.02% ammonium formate and acetonitrile. The total analysis time was 13.5 min. The three compounds were detected using electrospray ionisation in the positive mode. Standard curves were linear over the concentration range 5-5000 µg/L (r>0.997), bias was ≤ ±20%, intra- and inter-day coefficients of variation (imprecision) were <8% and the limit of detection was 5 µg/L. The limit of quantitation was set at 100 µg/L. Matrix effects were up to 140% but these were accounted for by the internal standard. The assay is being used successfully in clinical practice to enhance the safe and effective use of methylphenidate.

Determination of dexamethasone and dexamethasone sodium phosphate in human plasma and cochlear perilymph by liquid chromatography/tandem mass spectrometry.

A rapid, simple and sensitive liquid chromatography/tandem mass spectrometry (LC-MS/MS) assay was developed for the determination of dexamethasone (Dex) and dexamethasone sodium phosphate (Dex SP) in plasma and human cochlear perilymph. After proteins were precipitated with a mixture of acetonitrile and methanol, Dex, Dex SP and flumethasone, the internal standard, were resolved on a C18 column using gradient elution of 5 mM ammonium acetate and methanol. The three compounds were detected using electrospray ionisation in the positive mode. Standard curves were linear over the concentration range 0.5-500 µg/L (r>0.99), bias was <±10%, intra- and inter-day coefficients of variation (imprecision) were <10%, and the limit of quantification was 0.5 µg/L for both Dex and Dex SP. The assay has been used successfully in a clinical pharmacokinetics study of Dex and Dex SP in cochlear perilymph and plasma.

Determination of perhexiline and its metabolite hydroxyperhexiline in human plasma by liquid chromatography/tandem mass spectrometry.

Perhexiline is a drug that is used for treatment of moderate to severe angina pectoris that has not responded to other treatment. It has a low therapeutic index, and saturable metabolism that is also subject to genetic polymorphism (CYP2D6). Concentration monitoring of the parent drug and its major metabolite is considered necessary to optimise efficacy and reduce the risk of hepatotoxicity and neuropathy. A rapid, simple and sensitive liquid chromatography/tandem mass spectrometry (LC-MS/MS) assay was developed for the determination of perhexiline and its metabolite cis-hydroxyperhexiline in human plasma. After proteins were precipitated with acetonitrile, perhexiline, the major metabolite cis-hydroxyperhexiline and nordoxepin as the internal standard were resolved on a phenyl-hexyl column using gradient elution of 0.05% formic acid and methanol. The three compounds were detected using electrospray ionisation in the positive mode. Standard curves were linear over the concentration range 10-2000microg/L (r>0.999), bias was

Dissociation of neopterin and 7,8-dihydroneopterin from plasma components before HPLC analysis.

Measurement of plasma neopterin by HPLC with fluorescence detection is used clinically as a marker of immune cell activation in the management of a number of disease pathologies. HPLC analysis of neopterin requires the acidic removal of plasma proteins but we have found that 7,8-dihydroneopterin is oxidised to neopterin with varying yield. Using acetonitrile as the precipitant, we have measured substantially higher quantities of both total neopterin (7,8-dihydroneopterin and neopterin) and neopterin from plasma of healthy and septicemia patient's. Total neopterin concentrations were on average 50% and 200% greater in healthy and septicemia subjects, respectively, when measured after acetonitrile precipitation compared to trichloroacetic acid. Our data suggests that some pterin co-precipitates with proteins during acid treatment.

Determination of celecoxib in human plasma and breast milk by high-performance liquid chromatographic assay.

A rapid and simple HPLC assay was developed for the determination of celecoxib in human plasma and breast milk. After proteins were precipitated with acetonitrile, celecoxib was resolved on a C18 column and detected by UV detection at 254 nm. Standard curves were linear over the concentration range 10-2000 microg/L (r(2)>0.99). Bias was

Determination of rofecoxib in human plasma and breast milk by high-performance liquid chromatographic assay.

A rapid and simple HPLC assay was developed for the determination of rofecoxib in human plasma and breast milk. After solid-phase extraction, rofecoxib was resolved on a C18 column and detected by UV detection at 272 nm. Standard curves were linear over the concentration range 10-2000 microg/L (r2 >0.99). Intra- and inter-day coefficients of variation for both matrices were <10% and the limit of quantification was around 10 microg/L.

An enzyme-linked immunosorbent assay (ELISA) for methylphenidate (Ritalin ) in urine.

A direct enyzme-linked immunosorbent assay (ELISA) for urinary immunoreactive methylphenidate (Ritalin), in which a standard 96-well microtiter plate is used, is described. For this ELISA, a methylphenidate-thyroglobulin conjugate is immobilized to the microtiter plate and competes with methylphenidate in the standard or urine sample for antibody-binding sites. After washing, the sheep methylphenidate antibody bound to immobilized methylphenidate is detected with peroxidase-labelled goat antisheep IgG. Following a further wash, tetramethylbenzidine is added, color is developed, and the plate is read at 450 nm on an ELISA plate reader. This method is unaffected by drugs of abuse and is suitable for routine use in the toxicology laboratory.

Rapid and simple high-performance liquid chromatographic assay for the determination of metformin in human plasma and breast milk.

A rapid and simple high-performance liquid chromatographic (HPLC) assay for the determination of metformin in human plasma and breast milk is described. After proteins were precipitated with acetonitrile, metformin and the internal standard buformin were resolved on a cation-exchange column and detected by UV detection at 236 nm. Standard curves were linear over the concentration range 20.0-4000 microg/l. Intra- and inter-day coefficients of variation were <9.0% and the limit of quantification was around 20 microg/l.