Suspected poor-quality medicines in Kenya: a retrospective descriptive study of medicine quality-related complaints reports in Kenya's pharmacovigilance database.

Poor-quality, substandard and falsified, medicines pose a significant public health threat, particularly in low-middle-income countries. A retrospective study was performed on Kenya's Pharmacovigilance Electronic Reporting System (2014-2021) to characterize medicine quality-related complaints and identify associations using disproportionality analysis. A total of 2767 individual case safety reports were identified, categorized into medicines with quality defects (52.1%), suspected therapeutic failure (41.6%), and suspected adverse drug reactions (6.3%). Predominantly reported were antineoplastic agents (28.6%), antivirals (11.7%), and antibacterial agents (10.8%) potentially linked to non-adherence to good manufacturing practices, inappropriate usage and supply chain degradation. Notably, analgesics (8.2%), and medical devices (3.5%) notified had quality defects, predominantly from government health facilities (60.0%). Antineoplastic agents (20.2%) and antivirals (3.7%) were frequently reported from suspected therapeutic failures and suspected adverse drug reactions, respectively, across both private for-profit facilities (26.5%) and not-for-profit facilities (5.4%). Underreporting occurred in unlicensed health facilities (8.1%), due to unawareness and reporting challenges. Pharmacists (46.1%), and pharmaceutical technicians (11.7%) predominantly reported quality defects, while medical doctors (28.0%) reported suspected therapeutic failures. Orally administered generic medicines (76.9%) were commonly reported, with tablets (5.8%) identified as potential sources of suspected adverse drug reactions, while quality defects were notified from oral solutions, suspensions, and syrups (7.0%) and medical devices (3.9%). The COVID-19 pandemic correlated with reduced reporting possibly due to prioritization of health surveillance. This study provides valuable evidence to supporting the use of medicine quality-related complaints for proactive, targeted regulatory control of high-risk medicines on the market. This approach can be strengthened by employing standardized terminology to prioritize monitoring of commonly reported suspected poor-quality medicines for risk-based sampling and testing within the supply chain.

Synthesis and antitubercular activity of novel 4-arylalkyl substituted thio-, oxy- and sulfoxy-quinoline analogues targeting the cytochrome bc1 complex.

A library of 4-substituted quinolines was synthesised based on the structural features of the privileged 4-(benzylthio)-6-methoxy-2-methylquinoline scaffold. Quinoline-based chemical probes have proven to be effective anti-tuberculosis agents with the ability of inhibiting components of Mycobacterium tuberculosis (MTB) respiratory chain including the b subunit of the cytochrome bc1 complex. Novel 4-(arylalkyl)-thio, -oxy and sulfoxy-quinoline analogues were tested for their ability to inhibit the growth of MTB H37Rv and QcrB mutant strains, and the compounds mode of action was investigated. Members of the 4-subtituted thio- and sulfoxyquinoline series exhibited significant growth inhibitory activity in the high nanomolar range against wild-type MTB and induced depletion of intracellular ATP. These probes also showed reduced potency in the QcrB T313I mutant strain, thus indicating the cytochrome bc1 oxidase complex as the molecular target. Interestingly, new 4-(quinolin-2-yl)oxy-quinoline 4i was more selective for the QcrB T313I strain compared to the wild-type strain.

Hyphenated mass spectrometry techniques for assessing medication adherence: advantages, challenges, clinical applications and future perspectives.

Nonadherence to prescribed pharmacotherapy is an understated public health problem globally and is costing many patients their chance to return to good health and healthcare systems billions. Clinicians need an accurate assessment of adherence to medications to aid the clinical decision-making process in the event of poor patient progress and to maximise the patient health outcomes from the drug therapies prescribed. An overview of indirect and direct methods used to measure medication adherence is presented, highlighting the potential for accurate measuring of drugs in biological samples using hyphenated mass spectrometry (MS) techniques to provide healthcare professionals with a reliable evidence base for clinical decision making. In this review we summarise published applications of hyphenated MS techniques for a diverse range of clinical areas demonstrating the rise in the use of such direct methods for assessing medication adherence. Although liquid chromatography-tandem mass spectrometry (LC-MS/MS) methods using plasma, serum and urine samples are the most popular, in recent years increased attention has been given to liquid chromatography high-resolution mass spectrometry (LC-HRMS) methods and alternative biosample matrices including hair, saliva and blood microsamples. The advantages and challenges of using hyphenated MS techniques to address this healthcare problem are also discussed alongside future perspectives.

Utilizing quantitative dried blood spot analysis to objectively assess adherence to cardiovascular pharmacotherapy among patients at Kenyatta National Hospital, Nairobi, Kenya.

The burden of cardiovascular disease (CVD) is rising in Kenya and non-adherence to cardiovascular pharmacotherapy is a growing global public health issue that leads to treatment failure, an increased risk of cardiac events and poor clinical outcomes. This study assessed adherence to selected cardiovascular therapy medications among CVD patients attending outpatient clinics at Kenyatta National Hospital, Kenya by determining drug concentration(s) in patient dried blood spot (DBS) samples. Patients who had been taking one or more of the five commonly prescribed CVD medications (amlodipine, atenolol, atorvastatin, losartan, and valsartan) for at least six months were enrolled. Each patient completed a short questionnaire about their medication history and then provided a finger-prick blood spot sample from which drug concentrations were determined by liquid chromatography-high resolution mass spectrometry analysis. Two hundred and thirty-nine patients (62.3% female) participated in the study. The median number of medications used by patients was 2 (IQR 75%-25% is 3-1). Less than half (117; 49.0%) of patients were adherent to their prescribed CVD pharmacotherapy. Binary regression analysis revealed a significant correlation between non-adherence and the number of medications in the treatment regimen (Odds Ratio (OR) 1.583; 95%CI: 0.949-2.639; P-value = 0.039) and that gender was not an independent predictor of medication adherence (OR 1.233; 95%CI: 0.730-2.083; P-value = 0.216). Valuable information about adherence to each medication in the patient's treatment regimen was obtained using quantitative DBS analysis showing that adherence to CVD medications was not uniform. DBS sampling, due its minimally invasive nature, convenience and ease of transport is a useful alternative matrix to monitor adherence to pharmacotherapies objectively, when combined with hyphenated mass spectrometry analytical techniques. This information can provide physicians with an evidence-based novel approach towards personalization and optimization of CVD pharmacotherapy and implementing interventions in the Kenyan population, thereby improving clinical outcomes.

Aminobenzofuran-containing analogues of proximicins exhibit higher antiproliferative activity against human UG-87 glioblastoma cells compared to temozolomide.

A new series of proximicin analogues containing a benzofuran moiety as the replacement of the di-furan scaffold of the parent compound were synthesised and evaluated for their anti-proliferative activities against human glioblastoma cells U-87 MG. Proximicins A, B, and C are secondary metabolites produced by Verrucosispora Fiedleri MG-37, a Gram-positive actinomycete isolated from deep-sea sediment. Proximicins exhibit significant cytotoxic and apoptotic effects in a number of tumour cell lines, although further investigations on these natural products biological activity are hampered by the challenging synthesis of their constitutive di-furan unit. Therefore, the easily-synthesisable benzofuran ring was elected as a replacement of the di-furan platform, and a library of proximicin analogues was prepared in which different substituents were introduced at both the N-terminus and C-terminus of the benzofuran core unit. The novel compounds were tested against U-87 MG, as it was previously found that proximicins targeted this cancerous cell line, and the human healthy cell line WI-38. Temozolomide, the chemotherapeutic agent of choice for the treatment of glioblastoma, was used as a control. Analysis of growth inhibitory concentration values revealed that a number of furan-benzofuran-containing proximicin analogues, including 23(16) (IC50 U-87 MG = 6.54 µg mL-1) exhibited higher antiproliferative activity against glioblastoma cells compared to both proximicins A-C and temozolomide (IC50 U-87 MG = 29.19 µg mL-1) in U-87 MG.

Patients Access to Medicines - A Critical Review of the Healthcare System in Kenya.

Access to affordable, safe, effective, and quality-assured medicines by a patient is important for good health outcomes. Unfortunately, there is sparse literature published on the pharmaceutical enablers that may increase the sale of a substandard and falsified (SF) medicine to a patient in Kenya. The review highlights some of the factors that may facilitate the entry of SF medicines into the legitimate pharmaceutical supply chain and discusses their impact on patient access to medicines. Lack of essential medicines in public health facilities is an important factor that may contribute to increased demand for medicine-related out-of-pocket expenses from private health facilities, thus a likelihood for a patient purchasing SF medicine from unlicensed and illegal medicine outlets or unregulated websites. The need to increase medicine availability in the public sector by the Ministry of Health (MOH) is emphasized in addition to the strengthening of public procurement to cushion it from corruption and mismanagement. In addition, the MOH should promote local pharmaceutical manufacturing and implement a medicine pricing containment policy to avoid abuse and prevent overexploitation of patients, increase medicine price transparency, and reduce pharmaceutical supply chain distortion. Recommended regulatory reviews include accreditation of unlicensed illegal medicine outlets to facilitate accountability, regulatory oversight, and active surveillance. The national post-market surveillance regulatory capacity should be strengthened to improve rational medicine use. A 3-year diploma course should be replaced with a shorter 1- or 2-year pharmaceutical support staff training not eligible to superintend a pharmacy. The recommended legislative review includes a mandatory clause to enforce generic prescribing and the implementation of generic substitution by health workers. Unethical manipulative pharmaceutical marketing practices should carry stiffer penalties to deter malpractice. Future research areas include investigation of medicine prescribing and dispensing practices, medicine consumption studies, medicine price differences within different health sub-sectors, and between licensed pharmacies and unlicensed illegal medicine outlets.

Effect of varying molecular weight of dextran on acrylic-derivatized dextran and concanavalin A glucose-responsive materials for closed-loop insulin delivery.

AIM: Dextran methacrylate (dex-MA) and concanavalin A (con A)-methacrylamide were photopolymerized to produce covalently cross-linked glucose-sensitive gels for the basis of an implantable closed-loop insulin delivery device. METHODS: The viscoelastic properties of these polymerized gels were tested rheologically in the non-destructive oscillatory mode within the linear viscoelastic range at glucose concentrations between 0 and 5% (w/w). RESULTS: For each cross-linked gel, as the glucose concentration was raised, a decrease in storage modulus, loss modulus and complex viscosity (compared at 1 Hz) was observed, indicating that these materials were glucose responsive. The higher molecular weight acrylic-derivatized dextrans [degree of substitution (DS) 3 and 8%] produced higher complex viscosities across the glucose concentration range. CONCLUSIONS: These studies coupled with in vitro diffusion experiments show that dex-MA of 70 kDa and DS (3%) was the optimum mass average molar mass to produce gels that show reduced component leach, glucose responsiveness, and insulin transport useful as part of a self-regulating insulin delivery device.

Adherence to cardiovascular pharmacotherapy by patients in Iraq: A mixed methods assessment using quantitative dried blood spot analysis and the 8-item Morisky Medication Adherence Scale.

This study evaluated the adherence to prescribed cardiovascular therapy medications among cardiovascular disease patients attending clinics in Misan, Amara, Iraq. Mixed methods were used to assess medication adherence comprising the Arabic version of the eight-item Morisky Medication Adherence Scale (MMAS-8) and determination of drug concentrations in patient dried blood spot (DBS) samples by liquid chromatography-high resolution mass spectrometry. Three hundred and three Iraqi patients (median age 53 years, 50.5% female) who had been taking one or more of the nine commonly prescribed cardiovascular medications (amlodipine, atenolol, atorvastatin, bisoprolol, diltiazem, lisinopril, losartan, simvastatin and valsartan) for at least six months were enrolled. For each patient MMAS-8 scores were determined alongside drug concentrations in their dried blood spot samples. Results from the standardized questionnaire showed that adherence was 81.8% in comparison with 50.8% obtained using the laboratory-based microsample analysis. The agreement between the indirect (MMAS-8) and direct (DBS analysis) assessment approaches to assessing medication adherence showed significantly poor agreement (kappa = 0.28, P = 0.001). The indirect and direct assessment approaches showed no significant correlation between nonadherence to prescribed cardiovascular pharmacotherapy and age and gender, but were significantly associated with the number of medications in the patient's treatment regimen (MMAS-8: Odds Ratio (OR) 1.947, 95% CI, P = 0.001; DBS analysis: OR 2.164, 95% CI, P = 0.001). The MMAS-8 results highlighted reasons for nonadherence to prescribed cardiovascular pharmacotherapy in this patient population whilst the objective DBS analysis approach gave valuable information about nonadherence to each medication in the patient's treatment regimen. DBS sampling, due its minimally invasive nature, convenience and ease of transport is a useful alternative matrix to monitor adherence objectively in Iraq to cardiovascular pharmacotherapy. This information combined with MMAS-8 can provide clinicians with an evidence-based novel approach to implement intervention strategies to optimise and personalise cardiovascular pharmacotherapy in the Iraqi population and thereby improve patient health outcomes.

Glucose-sensitive gel rheology of dextran-concanavalin A mixtures suitable for self-regulating insulin delivery.

Aqueous concentrated plain mixtures of dextran and concanavalin A (con A) were examined for their rheological response to glucose for comparison with previously studied partially photopolymerized acrylic derivatives. Non-destructive oscillatory tests were undertaken within the linear viscoelastic range to examine the relationship between the rheometry and the stoichiometry of the interactive materials and to examine rheological parameters as affected by molecular weight, component ratio, temperature and glucose concentrations between 0 and 1% w/w. These simple formulations were studied at 1 and 10 Hz at 0.5% strain at both 20 and 37 degrees C. A second simplified rheological test was undertaken to demonstrate gel-sol reversibility and to produce a measure of equilibria created between these gels and glucose solutions with which they are in contact. This mimics the conditions in which the gel acts as a responsive gateway in the insulin delivery device. It proved that the gels equilibrate with glucose solutions, rather than indiscriminately removing glucose. This is important in terms of producing a delivery device that can respond in a reversible, glucose concentration-dependent manner. The method used for this is capable of relative values only but provides information not obtainable from conventional rheometry.

A review of Fourier Transform Infrared (FTIR) spectroscopy used in food adulteration and authenticity investigations.

The increasing demand for food and the globalisation of the supply chain have resulted in a rise in food fraud, and recent high profile cases, such as the Chinese milk scandal in 2008 and the EU horsemeat scandal in 2013 have emphasised the vulnerability of the food supply system to adulteration and authenticity frauds. Fourier Transform Infrared (FTIR) spectroscopy is routinely used in cases of suspected food fraud as it offers a rapid, easy and reliable detection method for these investigations. In this review, we first present a brief summary of the concepts of food adulteration and authenticity as well as a discussion of the current legislation regarding these crimes. Thereafter, we give an extensive overview of FTIR as an analytical technique and the different foods where FTIR analysis has been employed for food fraud investigations as well as the subsequent multivariate data analyses that have been applied successfully to investigate the case of adulteration or authenticity. Finally, we give a critical discussion of the applications and limitations of FTIR, either as a standalone technique or incorporated in a test battery, in the fight against food fraud.

Quantitative screening of the pharmaceutical ingredient for the rapid identification of substandard and falsified medicines using reflectance infrared spectroscopy.

The World Health Organization suggests that approximately 10% of medicines worldwide are either falsified or substandard with higher figures in low and middle income countries. Such poor quality medicines can seriously harm patients and pose a threat to the economy worldwide. This study investigates attenuated total reflectance-fourier transform infrared (ATR-FTIR) spectroscopy as a simple and rapid method for determination of drug content in tablet dosage forms. Paracetamol was used as the model pharmaceutical ingredient. Spectra of standard mixtures of paracetamol with different excipients formed the basis for multivariate PLS based quantitative analysis of simulated tablet content using different selected infrared absorbance bands. Calibration methods using ATR-FTIR were compared with the ATR-FTIR and conventional ultraviolet spectroscopic analyses of real tablet samples and showed that the paracetamol/microcrystalline cellulose mixtures gave optimum results for all spectral bands tested. The quantitative data for band 1524-1493cm-1 was linear (R2 ˃ 0.98; LOQ ≥ 10%w/w tablet). Global examples of paracetamol tablets were tested using this protocol and 12% of the tablet samples examined was identified as substandard. Each sample analysis was completed in just a few minutes. ATR-FTIR can therefore be used in the rapid screening of tablet formulations. The simplicity of the proposed method makes it appropriate for use in low and middle income countries where analytical facilities are not available.

Volumetric absorptive microsampling (VAMS) coupled with high-resolution, accurate-mass (HRAM) mass spectrometry as a simplified alternative to dried blood spot (DBS) analysis for therapeutic drug monitoring of cardiovascular drugs.

Here, volumetric absorptive microsampling (VAMS), used for the measurement of cardiovascular drugs, is compared against conventional dried blood spot (DBS) card sampling to evaluate adherence to prescribed medication. Volumetric absorptive microsampling (VAMS) is an attractive alternative to plasma sampling for routine drug monitoring and potentially overcomes haematocrit issues associated with quantitative bioanalysis of conventional dried blood spots. A quantitative VAMS-based LC-HRAM MS assay for atenolol, lisinopril, simvastatin and valsartan was developed and validated. The assay demonstrated acceptable linearity, selectivity, accuracy, precision, recovery and insignificant matrix effects with no impact of haematocrit on assay accuracy. Volunteers provided both VAMS and DBS 903 card samples (the current standard) to allow comparison of the two methods and demonstrate the potential utility of VAMS. Analysis of VAMS samples correctly identified drugs in volunteers known to be adherent, and found no false positives from volunteers known to be taking no medication. There was a strong correlation between the two sampling systems confirming the utility of VAMS. Therapeutic drug monitoring (TDM) can assist clinicians in deciding how to proceed with treatment in the event of poor improvement in patient health. VAMS could offer a potentially more efficient method of sample collection, with fewer rejected samples than the DBS approach.

Quantitative LC-HRMS determination of selected cardiovascular drugs, in dried blood spots, as an indicator of adherence to medication.

Dried blood spot (DBS) sampling was investigated as a means of obtaining micro-volume blood samples for the quantitative analyses of ten commonly UK prescribed cardiovascular drugs as an indicator of medication adherence. An 8mm disc was punched out from each DBS from calibration, quality control and volunteer samples and extracted using methanol containing the internal standard. Each extract was evaporated to dryness, the residue reconstituted in methanol:water (40:60v/v) containing 0.1% formic acid and analysed by LC-HRMS. Chromatography was performed using gradient elution on a Zorbax Eclipse C18 HD 100mm×2.1mm, 1.8µm pore size column with the column oven temperature at 40°C. Flow rate of the mobile phase was 0.6ml/min with a run time of 2.5min. Electrospray positive ionization was used for MS detection. Drug recoveries from spiked blood spots were 68% for simvastatin and ≥87% for all other target drugs. Compound specificity was obtained operating the MS with a 5ppm mass window. The LC-HRMS method was validated, with results for accuracy and precision within acceptable limits; analytes were stable at room temperature for at least 10 weeks and different blood spot volumes and haematocrit values had no significant effect. The LC-HRMS assay was used to analyse DBS samples from volunteers, some of whom were prescribed one or more of the target drugs. In results from 37 volunteers the assay successfully identified volunteers who were known to be either adherent or nonadherent; confirmed the correct drug/drugs for multiple prescriptions; demonstrated no false positives from other cardiovascular drugs; revealed several examples of unsuspected non-adherence. These results indicated that the developed assay was suitable for trials with patients.

The effect of degree of acrylic derivatisation on dextran and concanavalin A glucose-responsive materials for closed-loop insulin delivery.

Formulations of dextran methacrylate (dex-MA) and concanavalin A methacrylamide (con A-MA) were photo-polymerized to produce covalently cross-linked glucose-responsive materials for the basis of a closed-loop insulin delivery device. The viscoelastic properties of these polymerised materials were tested rheologically in the non-destructive oscillatory mode within the linear viscoelastic range at glucose concentrations between 0% and 5% w/w. The degree of acrylic substitution was varied for the dex-MA and con A-MA, and as the formulation glucose concentration was raised, a graded decrease in storage modulus, loss modulus and complex viscosity when compared at 1 Hz was observed for each cross-linked material. Increasing the degree of substitution (DS) of the derivatised dextran produced viscosity profiles at higher values throughout the glucose concentration range. A comparison with non-polymerised mixtures shows similar rheological properties but at much lower values across the chosen glucose concentration range. High-pressure liquid chromatography analyses and in vitro diffusion experiments showed that there were optimum degrees of derivatisation to minimise dex-MA and con A-MA component leach from the material. The in vitro diffusion experiments also showed that differential delivery of insulin in response to glucose was possible with candidate polymerised glucose-responsive formulations, thus highlighting the potential of such a novel glucose-sensitive material to be used as part of implantable closed-loop insulin delivery device.

Glucose-responsive UV polymerised dextran-concanavalin A acrylic derivatised mixtures for closed-loop insulin delivery.

A novel UV polymerised glucose-responsive mixture containing concanavalin A (con A) and dextran was synthesised and characterised as a "smart" biomaterial to form the basis of a closed-loop delivery device. Dextran and con A precursors were modified with acrylic side groups and then UV polymerised to produce covalently bonded mixtures which were examined by FTIR. The viscoelastic properties of these polymerised mixtures containing glucose concentrations between 0% and 5% w/w were also examined using oscillatory rheometry within the linear viscoelastic range across a frequency range of 0.01-50 Hz. As the formulation glucose concentration was raised, a graded decrease in storage modulus, loss modulus and complex viscosity when compared at 1 Hz was observed. Increasing the mixture irradiation time produced viscosity profiles at higher values throughout the glucose concentration range. The subsequent testing of such formulations in in vitro diffusion experiments revealed that the leaching of the mixture components is formulation dependent and is restricted significantly in the covalently bonded mixtures. Insulin delivery in response to glucose in the physiologically relevant glucose concentration range was demonstrated using the novel polymerised mixture at 37 degrees C. The performance of this covalently cross-linked glucose-responsive biomaterial has been improved in terms of increased mixture stability with reduced component leaching. This could, therefore be used as the basis of the design of a closed-loop drug delivery device for therapeutic agents used for the management of diabetes mellitus.

Development of a reversed-phase high-performance liquid chromatography method for the analysis of components from a closed-loop insulin delivery system.

A reversed-phase HPLC method has been developed which enables separation of the three components of a closed-loop insulin delivery system, namely concanavalin A methacrylamide (Con A-MA), dextran methacrylate (Dex-MA) and bovine insulin. The analysis of Con A-MA represents a significant challenge due to the formation of multiple conformations on contact with the chromatographic surface and the mobile phase. The extent of conformational change is shown to be dependent on a number of parameters: column temperature, mobile phase pH, contact time with the chromatographic surface, salt type and concentration and the organic modifier. By manipulation of these variables, protein denaturation can be minimised and recovery improved.

Rheological characterisation of dextran-concanavalin A mixtures as a basis for a self-regulated drug delivery device.

The rheological characterisation of glucose sensitive mixtures containing dextran and concanavalin A (con A) with and without glucose, was undertaken using oscillatory rheometry at 20 and 37 degrees C so that comparative data could be gathered in the linear viscoelastic (LVE) range. Measurements for a series of mixtures showed that complex viscosity is a function not only of the con A concentration but of the content and molecular weight of the dextran used. The extent of liquefaction on addition of glucose also depended on these factors. The tan delta profiles confirmed the change from semi-solid towards fluid behaviour. This occurs when glucose effects dismantling of the three-dimensional structure of the dextran-con A system by competitive binding to the glucose receptors in the protein. For the mixtures studied, the changes occurred between contents of 0 and 1% (w/w) glucose at 20 and 37 degrees C and form a useful basis for the formulation of a self-regulating delivery device for the control of hyper-and hypoglycaemia in diabetes.

Dried blood spot analysis to assess medication adherence and to inform personalization of treatment.

Little research using dried blood spot samples to assess adherence to medication has been reported. The World Health Organisation estimates that only half of the patients in the developed world take their medication as prescribed. Additional costs to the healthcare provider include wasted medicines, avoidable additional hospital visits and non-optimum patient care. There is little evidence of information concerning medication adherence being made available to inform clinical decision making. In this article we explore the potential of the dried blood spot sample collection methodology as a means of identifying medication adherence to facilitate medicines optimization for a range of disparate diseases. Furthermore, the opportunity to personalize healthcare for different patients by assessing the clinically necessary therapeutic level of the relevant drugs is highlighted.

Bisoprolol, ramipril and simvastatin determination in dried blood spot samples using LC-HRMS for assessing medication adherence.

The use of dried blood spot (DBS) collection cards was investigated for the quantification of three therapeutic drugs used in cardiovascular therapy for assessing medication adherence. A liquid chromatography-high resolution mass spectrometry (LC-HRMS) method was developed and validated for the determination of bisoprolol, ramipril and simvastatin. Whole blood spiked with target analytes was used to produce 30 µl blood spots on specimen collection cards. An 8mm disc was cut from the dried blood spot and extracted using methanol: water (70:30, v/v) containing the internal standard, atenolol. Extracts were vortexed, sonicated and then centrifuged. Gradient chromatographic elution was achieved using a Zorbax Eclipse C18 HD 100 mm × 2.1 mm, 1.8 µm pore size column and a mobile phase flow rate of 0.6 ml/min and the column oven temperature at 40 °C with a run time of 3 min. MS detection was carried out in electrospray positive ion mode for the three target drugs and for the IS. Drug recoveries from spiked blood spots were ≥ 92% for bisoprolol and ramipril and ~43% for simvastatin and the drugs were stable in DBS for at least 12 weeks. Validation of the LC-HRMS method showed good linearity and the accuracy (relative error) and precision (coefficient of variation) values were within the pre-defined limits of ≤ 15% at all concentrations. Matrix effects and the effects of different volumes of blood applied to the collection card were investigated. The LC-HRMS method successfully identified control volunteers who were known to be either adherent or non-adherent. There were no false positives from volunteers taking other cardiovascular drugs or from volunteers receiving no medication.

Quantitative determination of atenolol in dried blood spot samples by LC-HRMS: a potential method for assessing medication adherence.

The use of blood spot collection cards was investigated as a means of obtaining small volume samples for the quantification of therapeutic drugs for assessing medication adherence. A liquid chromatography-high resolution TOF mass spectrometry (LC-HRMS) method, based on the measurement at the accurate mass to charge ratio of the target analyte, was used to ensure specificity for atenolol in the dried blood spot (DBS) samples. A working method was developed and validated. For the preparation of DBS samples whole blood spiked with analyte was used to produce 30 µl blood spots on specimen collection cards. A 5mm disc was cut from the dried blood spot and extracted using methanol:water (60:40, v/v) containing the internal standard, atenolol-d(7). Extracts were vortexed, sonicated and then centrifuged. Gradient chromatographic elution was achieved using an Ascentis Express C18 100mm×2.1mm column and a mobile phase flow rate of 0.2 ml/min and the column oven temperature at 30 °C. MS detection was carried out in electrospray positive ion mode for target ions at accurate mass m/z 267.1703 for atenolol and 274.2143 for the IS. Drug extraction efficiency from spiked blood spots was demonstrated to be 96±5% and the drug was stable in DBS for at least 10 weeks. The developed LC-HRMS method was linear within the tested calibration range of 25-1500 ng/ml and validation showed the accuracy (relative error) and precision (coefficient of variation) values were within the pre-defined limits of ≤ 5% at all concentrations with a limit of quantification of 25 ng/ml. Factors with potential to affect drug quantification measurements such as the matrix effects, volume of blood applied onto the collection card and effect of different sampling cards were investigated. The developed LC-HRMS method was applied to blood spots on sampling card taken from adult healthy volunteers previously administered a 50mg atenolol tablet and a DBS concentration-time profile was obtained for atenolol. Requiring only a micro volume (30 µl) blood sample for analysis, the developed DBS based assay has the potential to assess patient adherence to atenolol.