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Metadrenaline Analysis Essay

Introduction

Section:

Phaeochromocytomas are neuroendocrine tumours that typically arise from catecholamine-producing chromaffin cells in the adrenal glands. Approximately 80–85% of phaeochromocytomas arise from the adrenal medulla.1 Clinical symptoms are largely due to the action of excess catecholamine secretion, which has provided the basis for biochemical assays for the diagnosis of phaeochromocytoma. Traditional methods use the measurement of the catecholamines – noradrenaline (norepinephrine), adrenaline (epinephrine) and dopamine, all of which are derived from tyrosine. The catecholamines are metabolized to normetadrenaline and metadrenaline by two mechanisms – catechol-o-methylation, using catechol-O-methyl transferase (COMT) and deamination by mitochondrial monoamine oxidase. COMT methylates the C-3 hydroxyl position of the benzene ring forming normetadrenaline, metadrenaline and 3-methoxytyramine from noradrenaline, adrenaline and dopamine, respectively. These are subsequently converted to vanillymandelic acid. Metadrenalines (normetadrenline and metadrenaline) exist in both free and conjugated forms. Total normetadrenaline is used here to mean the sum of free and conjugated normetadrenaline and total metadrenaline is used here to mean the sum of free and conjugated metadrenaline.

Abstract

Arterial hypertension is one of the most preventable causes of premature morbidity and mortality with resistant hypertension reported to be present in 5–30% of the total hypertensive population. Despite the poor prognosis, as many as 53% of those with resistant hypertension are reported to be nonadherent to their prescribed medication. An objective test of adherence, which is easy to administer, quick, inexpensive and reliable, is therefore needed to identify patients with true resistance to antihypertensive drugs to optimize their treatment. We have developed a novel LC–MS-MS method for the detection of 23 commonly prescribed antihypertensive medications in urine. The validated method was subsequently applied to the analysis of urine from a cohort of 49 individuals who were taking at least one antihypertensive agent in the screening profile to determine their adherence. The screening method was found to be reproducible, sensitive and specific with the limit of detection ranging from 0.1 to 1.0 µg/L. Sample preparation is rapid (30 s) and simple, with a total analysis time of 11 min. The assay successfully identified the majority of drugs our cohort had admitted to taking (88%) with drugs not detected in urine, potentially indicating nonadherence to prescribed medication. The performance of this simple, robust LC–MS-MS procedure is suitable for screening urine for the presence of commonly prescribed antihypertensive medications. The assay, which can easily be implemented in other laboratories, has the potential to significantly improve investigation and management of resistant hypertension.

Introduction

Arterial hypertension is one of the most preventable causes of premature morbidity and mortality in the world. The global prevalence of hypertension in adults was 26% in 2000 and is expected to go up to 29% in 2025 (1). It contributes to 62% of strokes and 49% of heart disease, leading to 7.1 million deaths a year, equivalent to 13% of all deaths in the world (2). Successful blood pressure (BP) lowering results in reductions of both morbidity and mortality (3).

Resistant hypertension is defined as BP above 140/90 mmHg while on ≥3 antihypertensive agents (one of which is usually a diuretic) at optimal or maximum tolerated doses (4–7). Patients with resistant hypertension are almost 50% more likely to experience a cardiovascular event compared with those without resistant hypertension (8). Resistant hypertension is reported to be present in 5–30% of the total hypertensive population (6). However, true prevalence of resistant hypertension is difficult to determine because of apparent resistance due to white coat hypertension, poor adherence with prescribed medication, poor BP measurement technique and inappropriate combination of treatment (5).

Poor medication adherence is common among patients with hypertension, with many studies reporting low adherence rates associated with inadequate BP control (9–13). As many as half of the patients labeled as having resistant hypertension have been reported to be nonadherent to prescribed medication (14). Our own experience from a directly observed therapy (DOT) clinic is similar, with suboptimal medication adherence being present in as many as half of the patients with resistant hypertension (15).

Despite the recognition that nonadherence is an important public health problem, the definitions of adherence vary and tests for adherence are imperfect (16). A number of indirect methods are commonly used to assess adherence including patient interviewing, prescription refill and pill counts. In the UK, most centers admit patients with resistant hypertension to hospital for supervised administration of medications and monitoring of BP. A few centers have established DOT clinics (15, 17). This method is costly in regard to bed/clinic usage and staff time, and inconvenient to patients. Therefore, there is a need for the development of an objective test of adherence, which is easy to administer, quick, inexpensive and reliable with a view to improving patient care by identifying those with true resistance to antihypertensive treatment. This will also allow research into areas including the factors affecting and tools designed to improve medication adherence (16).

Numerous techniques have been employed by laboratories to screen various biological matrices for drugs and metabolites. These techniques have included immunoassay, thin layer chromatography (TLC) and gas or liquid chromatography with or without mass spectrometry. For many years, gas chromatography with mass spectrometry (GC–MS) was the method of choice for toxicological analysis and is still of great value in the analysis of certain analytes, e.g., toxic alcohols (18). Owing to the superior specificity and sensitivity offered by liquid chromatography–tandem mass spectrometry (LC–MS/MS) coupled with the relative ease of sample preparation when compared with GC–MS, has seen LC–MS/MS adopted in many clinical laboratories for a number of biochemical and toxicological applications (19–22). Samples for LC–MS/MS analysis may still require some degree of sample preparation before analysis [for example, protein precipitation, liquid–liquid extraction or solid-phase extraction (SPE)] depending on the characteristics and concentration of the analyte in question. Recent improvements in the sensitivity of LC–MS-MS systems have led to the development of screening techniques, which allow samples to be simply diluted and then directly injected onto the analytical system (23). This offers a rapid sample preparation with minimal staff time and reagent cost when compared with other techniques, such as SPE, and has the advantage of screening for the broadest range of compounds as no one class of compound (e.g., opioids) is targeted in the extraction (c.f. liquid–liquid extraction).

Here, we present a novel ‘dilute-and-shoot’ LC–MS-MS method for the detection of a broad range of antihypertensive medications in human urine and show that the method is suitable for use in the investigation of resistant hypertension.

Materials and methods

Drug calibrators and internal standard preparation

Stock solutions of all drugs (Table I) were prepared by dissolving pure drug (Sigma, Poole, UK) in HPLC-grade methanol (Rathburn Chemicals Ltd, Walkerburn, UK) to give a concentration of 1.00 mg/mL. Ten calibration standards (1,000, 500, 250, 100, 50, 25, 10, 1, 0.5 and 0.1 µg/L) were prepared by appropriate dilution of the stock methanolic solutions in blank donor urine for all drugs measured. Internal quality controls (QCs) at three levels (5, 35 and 350 µg/L) were independently prepared in the same way.

Table I

Antihypertensive Agents Assayed in the Urine and their Primary Mode of Action

Class of drug Examples 
Calcium channel blockers (CCBs) Amlodipine, diltiazem, felodipine, verapamil, nifedipine 
Angiotensin-converting enzyme (ACE) inhibitors Lisinopril, perindopril, ramipril, enalapril 
Angiotensin receptor blockers (ARB) Losartan, irbesartan, candesartan 
Diuretics Indapamide, furosemide, bendrofluomethiazide, hydrochlorothiazide 
Sympathetic blockers (β- and α-blockers) Atenolol, labetalol, bisoprolol, doxazosin, metoprolol 
Others Spironolactone (aldosterone receptor antagonist), moxonidine (imidazoline receptor subtype 1 agonist) 
Class of drug Examples 
Calcium channel blockers (CCBs) Amlodipine, diltiazem, felodipine, verapamil, nifedipine 
Angiotensin-converting enzyme (ACE) inhibitors Lisinopril, perindopril, ramipril, enalapril 
Angiotensin receptor blockers (ARB) Losartan, irbesartan, candesartan 
Diuretics Indapamide, furosemide, bendrofluomethiazide, hydrochlorothiazide 
Sympathetic blockers (β- and α-blockers) Atenolol, labetalol, bisoprolol, doxazosin, metoprolol 
Others Spironolactone (aldosterone receptor antagonist), moxonidine (imidazoline receptor subtype 1 agonist) 

View Large

Deuterated amlodipine (amlodipine-d4), bisoprolol (bisoprolol-d5), doxazosin (doxazosin-d8), hydrochlorothiazide (hydrochlorothiazide 13C,d2), oxazepam (oxazepam-d5), morphine (morphine-d3) ramipril (ramipril-d5) and (±)-11-nor-9-carboxy-Δ9-THC (THC-COOH-d3) were used as internal standards (Cerilliant and LGC) at a working concentration of 100 µg/L in HPLC-grade water (Rathburn Chemicals) containing 0.1% formic acid and 1 mM ammonium formate (Sigma).

Sample preparation

Standards or samples (50 µL) were manually pipetted into a 1.1-mL screw-topped conical glass vial (Kinesis Solutions, Cambridgeshire, UK). To this, 150 µL of working internal standard was added. Samples were vortex-mixed for 10 s and transferred to the autosampler for analysis. About 20 µL of sample was injected and analyzed by LC–MS-MS.

Creatinine in urine was measured using the kinetic alkaline picrate method on the Abbott Architect c-8000 analyzer (Abbott Diagnostics, Abbott Park, IL, USA).

LC–MS-MS

The instrumentation consisted of a Shimadzu high-performance liquid chromatograph (Shimadzu, Milton Keynes, UK) and an API 4000 tandem mass spectrometer (AB Sciex, Warrington, UK) using an electrospray ionization ion source. The column used was a Hypersil Gold column (1.9 µm, 100 mm × 2.1 mm; Thermo Scientific, Hemel Hempstead, UK) with a Gemini C-18 Guard Column (4 × 3 mm; Phenomenex, Cheshire, UK) both maintained at 60°C. The mobile phases utilized were (A) HPLC-grade water containing 0.1% formic acid and 1 mM ammonium formate (Sigma) and (B) 90% HPLC-grade acetonitrile (Rathburn Chemicals) containing 0.1% formic acid and 1 mM ammonium formate. Gradient elution was employed for the analysis with the proportion of mobile phase B being maintained at 5% for 0.5 min, and then increased to 100% by 3.3 min and held at 100% for 0.7 min. Percentage mobile phase B was then reduced to 5% for 1 min giving a total run time of 5.5 min. The flow rate was 0.5 mL/min. The mass spectrometer parameters are given in Table II. To optimize the analysis of all drugs screened, each sample was run twice, once in positive ionization mode and once in negative ionization mode. Representative chromatographs at a standard concentration of 25 µg/L are shown in Figure 1.

Table II

Mass Spectrometer Settings for all Drugs Screened For

Drug Precursor ion (m/z) (ionization mode) Product ion (m/zCollision energy (V) Declustering potential (V) Dwell time (ms) Retention time 
Amlodipine 409.0 (+) 170.0 (Quant) 44.0 59.0 20 3.08 
208.0 (Qual) 38.0 56.0 20 
Atenolol 267.0 (+) 56.3 (Quant) 50.3 79.3 20 1.78 
144.9 (Qual) 36.0 82.0 20 
Bendroflumethiazide 421.1 (−) 329.0 (Quant) −37.0 −123.0 70 3.19 
290.0 (Qual) −32.0 −123.0 70 
Bisoprolol 326.0 (+) 91.0 (Quant) 25.9 91.0 20 2.72 
74.0 (Qual) 42.5 95.5 20 
Candesartan 441.0 (+) 192 (Quant) 39.7 86.8 20 3.18 
207 (Qual) 37.1 77.9 20 
Carenone (spironolactone) 341.0 (+) 107.0 (Quant) 41.0 128.0 50 3.61 
Diltiazem 414.0 (+) 178.2 (Quant) 33.9 86.8 20 2.90 
150.3 (Qual) 59.2 150.3 20 
Doxazosin 452.0 (+) 344.0 (Quant) 41.6 129.0 20 2.78 
247.0 (Qual) 55.6 131.5 20 
Enalapril 377.0 (+) 234.2 (Quant) 27.4 81.6 20 2.77 
160.0 (Qual) 38.4 81.6 20 
Felodipine 384.0 (+) 338.0 (Quant) 15.5 75.0 20 3.98 
324.0 (Qual) 34.4 75.8 20 
Furosemide 329.3 (−) 205.0 (Quant) −29.3 −65.8 70 2.93 
Hydrochlorothiazide 295.9 (−) 205.1 (Quant) −33.9 −81.7 70 1.86 
125.9 (Qual) −42.4 −118.4 70 
Indapamide 366.0 (+) 90.9 (Quant) 55.4 92.0 20 3.08 
117.0 (Qual) 58.0 86.0 20 
Irbesartan 429.0 (+) 207.0 (Quant) 34.0 103.0 20 3.32 
180.0 (Qual) 58.0 108.0 20 
Labetalol 329.0 (+) 161.9 (Quant) 35.9 80.4 20 2.61 
91.0 (Qual) 60.4 70.6 20 
Lisinopril 406.0 (+) 84.0 (Quant) 45.5 92.8 50 2.08 
Losartan 423.4 (+) 207.2 (Quant) 35.0 75.0 20 3.23 
180.0 (Qual) 55.0 108.2 20 
Metoprolol 268.0 (+) 116.0 (Quant) 25.5 115.0 20 2.43 
191.0 (Qual) 26.6 73.0 20 
Moxonidine 242.0 (+) 56.2 (Quant) 66.6 96.1 20 1.77 
136.1 (Qual) 42.9 57.9 20 
Nifedipine 347.0 (+) 254.2 (Quant) 25.7 71.0 20 3.39 
211.1 (Qual) 26.3 71.0 20 
Perindopril 369.0 (+) 172.3 (Quant) 30.0 75.0 50 2.87 
98.1 (Qual) 49.0 82.0 50 
Ramipril 417.0 (+) 117.2 (Quant) 55.0 78.0 50 3.07 
130.2 (Qual) 43.0 78.0 50 
Verapamil 455.0 (+) 303.4 (Quant) 37.0 116.0 20 3.08 
164.8 (Qual) 34.9 116.0 20 
Amlodipine-d4413.0 (+) 170.0 44.0 59.0 20 3.08 
Bisoprolol-d5331.0 (+) 74.0 42.5 95.5 20 2.72 
Doxazosin-d8458.6 (+) 351.4 43.2 118.2 20 2.78 
Hydrochlorothiazide 13C,d2 299.0 (−) 77.8 −45.6 −117.2 20 1.86 
Morphine-d3289.0 (+) 153.0 57.2 114.6 20 1.08 
Oxazepam-d5292.1 (+) 246.2 31.6 108.0 20 3.14 
Ramipril-d5421.6 (+) 121.4 56.2 95.6 20 3.07 
THC-COOH-d3346.1 (−) 302.0 −28.7 −148.7 30 4.17 
Drug Precursor ion (m/z) (ionization mode) Product ion (m/zCollision energy (V) Declustering potential (V) Dwell time (ms) Retention time 
Amlodipine 409.0 (+) 170.0 (Quant) 44.0 59.0 20 3.08 
208.0 (Qual) 38.0 56.0 20 
Atenolol 267.0 (+) 56.3 (Quant) 50.3 79.3 20 1.78 
144.9 (Qual) 36.0 82.0 20 
Bendroflumethiazide 421.1 (−) 329.0 (Quant) −37.0 −123.0 70 3.19 
290.0 (Qual) −32.0 −123.0 70 
Bisoprolol 326.0 (+) 91.0 (Quant) 25.9 91.0 20 2.72 
74.0 (Qual) 42.5 95.5 20 
Candesartan 441.0 (+) 192 (Quant) 39.7 86.8 20 3.18 
207 (Qual) 37.1 77.9 20 
Carenone (spironolactone) 341.0 (+) 107.0 (Quant) 41.0 128.0 50 3.61 
Diltiazem 414.0 (+) 178.2 (Quant) 33.9 86.8 20 2.90 
150.3 (Qual) 59.2 150.3 20 
Doxazosin 452.0 (+) 344.0 (Quant) 41.6 129.0 20 2.78 
247.0 (Qual) 55.6 131.5 20 
Enalapril 377.0 (+) 234.2 (Quant) 27.4 81.6 20 2.77 
160.0 (Qual) 38.4 81.6 20 
Felodipine 384.0 (+) 338.0 (Quant) 15.5 75.0 20 3.98 
324.0 (Qual) 34.4 75.8 20 
Furosemide 329.3 (−) 205.0 (Quant) −29.3 −65.8 70 2.93 
Hydrochlorothiazide 295.9 (−) 205.1 (Quant) −33.9 −81.7 70 1.86 
125.9 (Qual) −42.4 −118.4 70 
Indapamide 366.0 (+) 90.9 (Quant) 55.4 92.0 20 3.08 
117.0 (Qual) 58.0 86.0 20 
Irbesartan 429.0 (+) 207.0 (Quant) 34.0 103.0 20 3.32 
180.0 (Qual) 58.0 108.0 20 
Labetalol 329.0 (+) 161.9 (Quant) 35.9 80.4 20 2.61 
91.0 (Qual) 60.4 70.6 20 
Lisinopril 406.0 (+) 84.0 (Quant) 45.5 92.8 50 2.08 
Losartan 423.4 (+) 207.2 (Quant) 35.0 75.0 20 3.23 
180.0 (Qual) 55.0 108.2 20 
Metoprolol 268.0 (+) 116.0 (Quant) 25.5 115.0 20 2.43 
191.0 (Qual) 26.6 73.0 20 
Moxonidine 242.0 (+) 56.2 (Quant) 66.6 96.1 20 1.77 
136.1 (Qual) 42.9 57.9 20 
Nifedipine 347.0 (+) 254.2 (Quant) 25.7 71.0 20 3.39 
211.1 (Qual) 26.3 71.0 20 
Perindopril 369.0 (+) 172.3 (Quant) 30.0 75.0 50 2.87 
98.1 (Qual) 49.0 82.0 50 
Ramipril 417.0 (+) 117.2 (Quant) 55.0 78.0 50 3.07 
130.2 (Qual) 43.0 78.0 50 
Verapamil 455.0 (+) 303.4 (Quant) 37.0 116.0 20 3.08 
164.8 (Qual) 34.9 116.0 20 
Amlodipine-d4413.0 (+) 170.0 44.0 59.0 20 3.08 
Bisoprolol-d5331.0 (+) 74.0 42.5 95.5 20 2.72 
Doxazosin-d8458.6 (+) 351.4 43.2 118.2 20 2.78 
Hydrochlorothiazide 13C,d2 299.0 (−) 77.8 −45.6 −117.2 20 1.86 
Morphine-d3289.0 (+) 153.0 57.2 114.6 20 1.08 
Oxazepam-d5292.1 (+) 246.2 31.6 108.0 20 3.14 
Ramipril-d5421.6 (+) 121.4 56.2 95.6 20 3.07 
THC-COOH-d3346.1 (−) 302.0 −28.7 −148.7 30 4.17 

View Large

Figure 1.

Representative chromatographs from a standard containing all drugs at a concentration of 25 µg/L run in positive (A) and negative (B) ionization modes. This figure is available in black and white in print and in color at JAT online.

Figure 1.

Representative chromatographs from a standard containing all drugs at a concentration of 25 µg/L run in positive (A) and negative (B) ionization modes. This figure is available in black and white in print and in color at JAT online.

Method validation

Selectivity

Drug-free urine samples from six healthy volunteers and a drug-free urine sample fortified with a mixture of commonly prescribed medications (Box 1) were processed and analyzed to test the selectivity of the method. The lack of peaks at retention times expected for analytes indicated acceptable selectivity and an absence of interfering substances in the urine.

Box 1

Commonly Prescribed and Over-The-Counter Medications Fortified into Blank Donor Urine to Test Specificity of Method.

Amitriptyline, brompheniramine, caffeine, carbamazepine, citalopram. clomipramine, codeine, cyclizine, desmethylcitalopram, desmethyltramadol, diazepam, dihydrocodeine, diphenhydramine, dothiepin, fluoxetine, gabapentin, glibenclamide, gliclazide, glipizide, ibuprofen, lamotrigine, levetiracetam, mirtazepine, morphine, naproxen, nordiazepam, nortriptyline, olanzapine, oxycodone, paracetamol, paroxetine, pethidine, phenobarbitone, pregabalin, promethazine, quetiapine, quinine, salicylate, sildenafil, theophylline, tolbutamide, trazodone, venlafaxine and zopiclone.

Linearity, lower limit of detection and lower limit of quantitation

Calibration standards at nine concentration levels were freshly prepared as outlined above. The linearity of the assay was calculated by a least-squares linear regression analysis of the peak area ratios of analyte to internal standard versus nominal analyte concentration.

The regression parameters of slope, intercept and correlation coefficient were calculated by the weighting factor, 1/x2. Linearity was assessed separately in triplicate using the coefficient of determination (r2) and by determining the error between nominal and measured concentration for each point on the calibration curve. An acceptable r2 value was deemed to be >0.95.

The area of blank urine sample was no more than 20% of lower limit of quantitation (LLOQ), where LLOQ was defined as the lowest detectable analyte concentration for which the values of precision (relative standard deviation, RSD) and accuracy (relative error, RE) were ≤20% and the signal-to-noise (S/N) ratio was ≥30. The error was evaluated by precision (RSD) and accuracy (RE) values, which were no greater than 20% for the other seven concentrations in the calibration curve. RE was calculated using RE (%) = [(measured concentration − nominal concentration)/nominal concentration] × 100. Lower limit of detection (LLOD) was defined as the analyte concentration with an S/N ratio of >10.

Precision, accuracy, recovery and matrix effect

QC urine samples were prepared as outlined above and were analyzed on the same day, and over three consecutive days, to evaluate precision and accuracy. Standard curves for each batch were prepared and analyzed on the same day to calculate the concentration of each QC sample. RSD and RE were calculated to estimate precision and accuracy. Recoveries were estimated at a QC concentration relevant for the linear range for each drug measured. The total recovery was evaluated by comparison of the concentration of drug obtained when either a standard amount of a solution containing known amount of drug or a water blank was added to an analytical standard, in a set of five separate experiments. Formula used was: Recovery (%) = [(measured concentration of analyte in fortified sample − measured concentration of analyte in diluted sample)/concentration of spike] × 100.

The matrix effect was assessed in six separate experiments at a relevant QC concentration by comparing the concentration of drug measured when the material was made either in HPLC-grade water or drug-free donor urine. Mean percentage difference between the two should ideally be <10% and statistically insignificant when the replicates are compared using a paired t-test. Statistical analysis was performed using Analyse-it.

Stability

The stability of the analyte in urinary samples was analyzed at 1–3 QC levels in triplicate after 24 h at room temperature (25°C), after 7 days at 4°C, after three complete freeze–thaw cycles from −20 to 25°C and after long-term storage at −20°C (28 days). A sample was considered to be stable in the biological matrix when the calculated concentrations were 80–120% of those of the freshly prepared samples.

Clinical effectiveness of the method

The method was applied to the analysis of random urine samples collected from a cohort of patients attending the hypertension clinic at Birmingham Heartlands Hospital. The patients volunteered to give a urine sample and provided a list of all prescribed medication taken in the 24 h prior to clinic attendance. The medication prescribed, dose taken and time of administration (where available) were recorded on the request form. Urine samples were transported to the laboratory within 6 h of collection and stored at −20°C until analysis. For drugs where two transitions are defined (quantifier and qualifier, Table II), both transitions had to be present at the correct retention time (within 0.2 min when compared with an analytical standard and the internal standard used) in order for the specimen to be positive to the drug in question. For those drugs where only one transition could be defined (furosemide, lisinopril and carenone), the presence of this transition at the correct retention time constituted a positive result. Transition ratios were not used for identification. In cases where concentrations of drug were above the linear range of the method, samples were diluted in blank donor urine to a concentration within the linear range, as suggested by the estimated concentration obtained. Samples were then reanalyzed. Drugs identified, and the concentration of these drugs in urine, are summarized in Tables VII and VIII.

Results

The 23 commonly used antihypertensive drugs belonging to various classes (Table I) were separated based on a retention time and MS–MS fragmentation. Although total chromatographic separation was not achieved in positive mode, all compounds could be separated based on drug-specific MS–MS fragmentation with no interference observed between drugs. Owing to the characteristics of the drugs under investigation, and to ensure the optimal sensitivity of the method, samples were run twice, once in positive mode and once in negative mode (Figure 1). Each run was 5.5 min in length, giving a total run time of 11 min per sample.

Selectivity

Typical chromatograms for all drugs screened for are depicted in Figure 1. No interfering signals were observed from the blank donor urine samples tested, or from the urine fortified with drugs listed in Box 1. Retention times were within 0.2 min in each case when compared with an analytical standard and the relevant internal standard.

Linearity, LLOD and LLOQ

Linearity, LLOQ and LLOD for all drugs are presented in Table III. Felodipine could be reliably detected with an LLOD of 10 µg/L; however, accurate quantitation of the drug could not be performed due to unacceptable RSD and RE when assessed for linearity.

Table III

Linearity and Recovery Results for All Drugs Screened For

Drug LLOD LLOQ ULOQ r2Recovery (%) 
Amlodipine 25 250 0.985 

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