Validation of SPE-HPLC determination of 1,4-benzodiazepines and metabolites in blood plasma, urine,...

Mohammad Nasir UddinVictoria F. SamanidouIoannis N. Papadoyannis

Laboratory of AnalyticalChemistry, Department ofChemistry, Aristotle University ofThessaloniki, Thessaloniki,Greece

Original Paper

Validation of SPE-HPLC determination of1,4-benzodiazepines and metabolites in bloodplasma, urine, and saliva

A simple, sensitive, selective, and reproducible RP-HPLC method with DAD detectionat 240 nm was developed for the determination of six 1,4-benzodiazepines: brom-azepam (BRZ), clonazepam (CLZ), diazepam (DZP), flunitrazepam (FNZ), lorazepam(LRZ), alprazolam (APZ); and two metabolites: a-hydroxyalprazolam (HALZ) and a-hydroxytriazolam (HTZL) in human plasma, urine, and saliva, using colchicine asinternal standard, after SPE using Nexus Varian cartridges. Separation was perform-ed on a Kromasil C8 (250 mm65 mm, 5 lm) analytical column with a gradientmobile phase containing methanol, ACN and 0.05 M ammonium acetate. Linearitywas held within the range 0.3–20.0 ng/lL, with coefficients of determination (r2)better than 0.997. The within- and between-day assay RSD at 2, 4, 8 ng/lL rangedfrom 0.03 to 4.7% and 0.5 to 7.0%, respectively in standards, from 1.3 to 7.9% and3.3 to 7.3%, respectively in plasma, from 2.1 to 6.0% and 2.1 to 7.8%, respectively inurine and at 0.5, 1.0, 2.0 ng/lL ranged from 2.22 to 5.8% and 2.2 to 8.1%, respec-tively, in saliva. The mean relative recoveries were 96.3–108.6, 96.0–108.2, 94.3–107.1, 97.0–107.0% in within-day assay and 96.8–107.7, 94.6–107.6, 93.2–105.8,96.0–108.6 in between-day assay for standard, plasma, urine, and saliva, respec-tively. The LOD and LOQ were 0.02–0.47 and 0.07–1.57 ng/lL, respectively.

Keywords: Benzodiazepines / Blood plasma / High-performance liquid chromatography / Metabo-lites / Solid-phase extraction / Urine and saliva /

Received: June 12, 2008; revised: August 18, 2008; accepted: August 19, 2008

DOI 10.1002/jssc.200800342

1 Introduction

Benzodiazepines (BDZs) constitute a large and importantclass of pharmaceutical active drugs with antiepileptichypnotic, tranquillizing, anticonvulsant, sedative,muscle relaxant, and amnesic properties. Thirty-five ofthem are controlled by the United Nations convention(1971), and more than 50 are available throughout theworld [1–3]. Since the introduction of the first 1,4-benzo-diazepine, chlordiazepoxide [3, 4], in 1960, they havebecome the drugs of choice for the treatment of anxiety,sleep disorders, status epilepsy, insomnia, frequent noc-turnal or early morning awakenings and other convul-sive disorders [5, 6]. Because of these properties they are

also frequently encountered in clinical and forensic case-work samples involving road traffic offences and/or drugoverdoses [2].

Several analytical methods have been published forthe quantification of BDZs and/or their metabolite(s) inhuman (or animal) biological fluids or tissue organextracts based on acid–base titration [7], polarography[8], potentiometry [9], fluorimetry [10], spectrophotome-try [11], MEKC (MECC) [12], adsorptive-stripping voltam-metry [13], differential pulse voltammetry (DPV) [14], TLC[15], immunoassay [16], RIA [17], and flow injection anal-ysis (FIA) [18]. By far most of the published methods use-ful in clinical and forensic laboratories are applyingchromatography. These include GC with electron cap-ture detection (GC-ECD) [19, 20], nitrogen–phosphorusdetection (GC-NPD) [20, 21], MS with negative ion chemi-cal ionization (GC-MS/NICI) [22] or SIM (GC-MS/SIM) [23],GC/MS/MS [24], and HPLC methods with several detectionmodes including UV (HPLC-UV) [25] or diode array detec-tor (HPLC-DAD) [26, 27], mass spectrometric detection[28] or HPLC/MS/MS [29]. HPLC/LC-MS or MS/MS interfacedwith ESI or atmospheric pressure chemical ionization(APCI) further improves the specificity and sensitivity of

Correspondence: Professor Ioannis N. Papadoyannis, Laboratoryof Analytical Chemistry, Department of Chemistry, AristotleUniversity of Thessaloniki, Thessaloniki, GR-541 24, GreeceE-mail: [email protected]: +30-2310997719

Abbreviations: APZ, alprazolam; BDZ, benzodiazepine; BRZ,bromazepam; CLZ, clonazepam; DAD, diode array detector;DZP, diazepam; FNZ, flunitrazepam; HALZ, a-hydroxyalprazo-lam; HTZL, a-hydroxytriazolam; LRZ, lorazepam

i 2008 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.jss-journal.com

3704 M. N. Uddin et al. J. Sep. Sci. 2008, 31, 3704 – 3717

J. Sep. Sci. 2008, 31, 3704 –3717 Liquid Chromatography 3705

detection and has emerged as a selective analyticalmethod in drug analysis [30–33].

However, each of these methods has its shortcomingssuch as lack of selectivity, inadequate sensitivity, use oflarge sample volumes (F1 mL) or use of expensive SPEcartridges. GC-MS is an excellent method that providesunambiguous identification of compounds with goodsensitivity; but needs an expensive instrument, time-con-suming sample clean-up thus increasing the complexity;suffers from the thermal instability at high column tem-perature used and low volatility of many BDZs requiringa derivatization step [33]. Though the combination of MSoffers the advantage of the separation power of HPLCwith its sensitivity and specificity, the technique involvesexpensive equipment, which is not affordable for mostnonresearch laboratories, particularly those in resource-poor countries.

Again, an effective sample pretreatment procedure,which removes interfering endogenous components andpreconcentrates the analytical compounds prior to HPLCseparation is often necessary to improve sensitivity.Either liquid–liquid extraction [34], SPE [26, 27, 30], on-line SPE [32], or solid-phase microextraction [35] was usu-ally employed, which could enrich the analytes by sev-eral folds, even 1–2 orders of magnitude. An alternativesample pretreatment method involves protein removalby precipitation (with organic solvent) and centrifuga-tion. The isolated supernatants could then be directlyintroduced into HPLC [36, 37] for determination. Owing

to lack of a preconcentration step, the direct supernatantintroduction technique could not provide the sensitivityhigh enough for pharmacokinetic study of the drug.However, SPE is still being exploited by the researchersfor attaining objectives, like reduced solvent consump-tion and disposal, reduced time, cost, and labor provid-ing higher concentration factors than LLE. It also pro-vides greater opportunity for selective isolation, such asfractionation of the sample into different compounds orgroups of compounds [38]. To the best of our knowledgeonly one paper has reported [39] applying SPE for the iso-lation of BDZs from saliva.

The present paper describes the development and vali-dation of a relatively simple, sensitive, and selectiveHPLC-DAD method for the determination of six 1,4-ben-zodiazepines including two metabolites as shown in Fig.1, in human plasma, urine, and saliva samples. The gra-dient elution provided a good separation of analytes. SPEwas used for sample pretreatment yielding extracts freefrom endogenous interference.

2 Experimental

2.1 Instrumentation

A Shimadzu (Kyoto, Japan) quaternary low-pressure gra-dient system was used for the chromatographic determi-nation of the examined analytes. The solvent lines weremixed in an FCV-10ALVP mixer. An LC-10ADVP pump


Figure 1. Chemical structures ofBDZs and metabolites.


equipped with a Shimadzu SCL-10ALVP System Control-ler, permitting fully automated operation, was used todeliver the mobile phase to the analytical column. Sam-ple injection was performed via a Rheodyne 7725i injec-tion valve (Rheodyne, Cotati, California, USA) equippedwith a 20 lL loop. Detection was achieved by an SPD-M10AVP Photodiode Array Detector, complied with dataacquisition software Lab Solutions-LC-Solutions by Shi-madzu. Degassing of the mobile phase was performed byhelium sparging in the solvent reservoirs by a DGU-10Bdegassing unit.

The analytical column, a Kromasil C8 (250 mm64mm, 5 lm) was purchased from MZ-Analysentechnik(Mainz, Germany). A glass vacuum-filtration apparatusobtained from Alltech Associates was employed for thefiltration of the buffer solution, using 0.2 lm cellulosenitrate 0.2 lm-WCN Type (47 mm DIA) membrane filters(Whatman Laboratory Division, Maidstone, England).The SPE study was carried out on a 12-port vacuum mani-fold from Supelco (Bellefonte, PA, USA). LC-18 cartridges500 mg/3 mL and DSC-18 500 mg/3 mL were suppliedfrom Supelco, Abselut Nexus (30 mg/1 mL) from Varianand Lichrolut RP-select B (200 mg/3 mL) from Merck. Allevaporations were performed with a Supelco 6-port Mini-Vap concentrator/evaporator.

2.2 Chemicals and reagents

Colchicine (95% HPLC) used as the internal standard wassupplied by Sigma (Sigma–Aldrich Chemie BV, The Neth-erlands). HPLC-grade organic solvents (methanol, ACN)were supplied by Carlo Erba (Milano, Italy). Water usedthroughout the study was purified as required by thereverse osmosis method to gain high-purity water with aMilli-Q water purification system from Millipore (Milli-pore, Bedford, MA, USA). Ammonium acetate p.a. wassupplied from Riedel-de Haen (Buchs, SG, Switzerland).Six drug standards and two metabolites were from SigmaChemical Company (St. Louis, MO, USA) and Biomol (Ger-many), respectively. Purity of reference compounds wasnot less than 98%.

2.3 Collection of specimens

Drug free plasma samples were kindly provided from theBlood Donation Unit of a State Hospital, while urine andsaliva samples were provided by one male healthy volun-teer. Pooled samples were prepared. Both urine and sal-iva samples were collected in polypropylene tube. Salivasamples were collected over 180 min by spitting at fixedtime intervals, without any stimulation of the saliva pro-duction, to obtain approximately 5 mL of sample. By the30 min period before sampling no food or drink wastaken and the mouth was rinsed with fresh water beforespitting [25, 26]. Supplied drug-free plasma and as soon

as possible after collection without any preservative theurine and the supernatant of saliva obtained after centri-fugation at 2500 g for 10 min to remove potential fooddebris, were separated into 1.5 mL aliquots in eppendorfand stored at –208C until analysis (performed within15 days).

2.4 Chromatographic conditions

Chromatography was performed under gradient condi-tions at ambient temperature (about 258C). The mobilephase consisted of a mixture of methanol, ACN, andammonium acetate buffer (0.05 M) at initial compositionof 14:32:54 by volume delivered in gradient mode asshown in Table 1 at a flow rate of 1.1 mL/min, generatingan operating backpressure of initially 230 bar, whichremained unchanged up to 14 min and finally reducedto 180 bar at 16 min. Before use, the mobile phase wasdegassed for 10 min with helium. The column effluentwas monitored with a variable wavelength UV–Vis detec-tor set at 240 nm.

2.5 Standard stock solutions

Stock solutions (100 ng/lL) of BDZs, metabolites and col-chicine (IS) were prepared by dissolving an appropriateamount of each compound in methanol and were storedat 48C, protected from light and used within 3 months.The stock solutions of drugs were further diluted dailybefore analysis with methanol to make seven workingmixture solutions (controlled solution) at concentrationsof 0.20–20.00 lg/mL for each compound containinginternal standard at a constant concentration of 4 ng/lL.A 0.05 M aqueous solution of ammonium acetate wasprepared by mixing the appropriate weight in Milli-Qwater and filtered before use.

2.6 Optimization of SPE protocol

SPE protocols were optimized in terms of recovery stud-ies of BDZs prior to the application to biological fluids.Various extraction protocols were tested to isolate theanalytes from samples using different sorbents or mix-


Table 1. Gradient program for chromatographic conditions ofproposed method

Time Mobile phase composition (%)

CH3OH CH3CN 0.05 M CH3COONH4

0.01 14 32 549.00 13 32 55

11.00 11 28 6112.00 40 28 32


ture of solvents at different compositions for elution ofthe adsorbed analytes. Extraction yield was estimated bythe comparison of peak area ratios of extracted standardsolutions versus nonextracted solutions.

2.7 Sample preparation

2.7.1 Preparation of spiked samples

Experimental standard stock solution (200 lL) at sevenconcentration levels of 0.2–20 ng/lL for spiked samplesor 200 lL of methanol for blank samples was added to ali-quots of 50 lL of pooled plasma or 100 lL of urine or500 lL saliva samples.

2.7.2 Sample pretreatment

Prior to extraction, biological fluids were pretreated forthe removal of proteins by precipitation with the addi-tion of 200 lL of ACN to spiked samples; to aliquots of50 lL of pooled blood plasma or 100 lL of urine or 500 lLsaliva samples (if necessary) or spiked blank samples.After centrifugation for 15 min at 3000 rpm, the clearsupernatant was applied to SPE cartridges.

2.7.3 SPE

Prior to extraction SPE cartridges were conditioned with2 mL of methanol and 2 mL of water. Subsequentlyspiked samples were applied after addition of 2 mL dis-tilled water to decrease the percentage content of ACN ofsupernatants. After washing with 1 mL water retaineddrugs were eluted with (261) mL MeOH/CH3CN (50:50)followed by the evaporation to dryness under a gentleflow of N2 at 308C. The residue was reconstituted with200 lL of methanol prior to their injection into theliquid chromatographic system or the tubes were closedbefore storing at –208C. Aliquots of 20 lL were injectedonto column.

2.8 Method validation

The developed method was validated in terms of ICH ana-lytical performance parameters like (CPMP/ICH/281/95,Note for guidance on validation of analytical procedures:Methodology, ICH topic Q2B validation of analytical pro-cedures: Methodology, Step 4 (CPMP Adopted December96)) precision, accuracy, selectivity, LOD, LOQ, linearity,system suitability, robustness, and stability.

3 Results and discussion

3.1 Chromatography

An RP HPLC procedure with DAD detection has beendescribed for the selective, sensitive, accurate, and repro-ducible quantitative analysis of BDZs and two metabo-lites in human biological samples. In compromisingpeak resolution and retention time, the mentioned con-ditions were accepted when the examined analytes werewell resolved providing better separation. The separationof BDZs and the internal standard was achieved in lessthan 16 min. Retention times of the examined BDZs are3.92 l 0.06 for the internal standard, 5.47 l 0.06 forbromazepam (BRZ), 7.17 l 0.11 for a-hydroxytriazolam(HTZL), 7.66 l 0.11 for hydroxyalprazolam (HALZ),9.00 l 0.13 for clonazepam (CLZ), 9.55 l 0.15 for flunitra-zepam (FNZ), 10.17 l 0.0.19 for lorazepam (LRZ),10.77 l 0.21 for ALP, and 15.77 l 0.12 min for diazepam(DZP). A representative chromatogram of extracted blankand spiked human plasma, urine, and saliva, obtainedunder the described chromatographic conditions areillustrated in Figs. 2–4.

3.2 Column efficiency

Retention factor (k = tR – t0/t0), separation factor (a = tR2 –t0/tR1 – t0), resolution factor Rs = 2(tR2 – tR1)/(w2 + w2), num-


Table 2. Study of column efficiency

Analytes Retentionfactor

Separationfactor

Resolutionfactor

No. of theoreticalplates

Asymmetryfactor

k = (tR – t0) a = k2 –k1 Rs = 2(tR2 – tR1)/(w1 + w2) N = 16(tR/w)2 AsF = b0.1/a0.11

IS 1.1 – – 1 228.2 1.8BRZ 2.0 1.8 3.2 1 577.0 1.8HTRZ 2.9 1.4 3.5 5 449.1 1.5HALZ 3.1 1.1 1.2 6 171.4 1.3CLZ 3.8 1.2 3.2 6 501.3 1.0FNZ 4.1 1.1 1.2 7 324.9 1.0LRZ 4.4 1.1 1.3 8 246.6 1.0ALZ 4.8 1.1 1.3 9 188.4 1.0DZP 7.5 1.6 13.1 35 656.3 1.5


ber of theoretical plates N = 16(tR/w)2 and asymmetry fac-tor (AsF = b0.1/a0.1) have been calculated to check the col-umn efficiency. Here, t0, tR1, and tR2 are the retention

times and w1 and w2 the baseline peak widths of succes-sive peaks. Results given in Table 2 show the excellentperformance of the column.


Figure 2. Chromatogram of (A) blank plasma and (B) plasma spiked with BDZs (4 ng/lL) under the conditions described in text.Peaks are as follows: IS (3.942 min), BRZ (5.551 min), HTRZ (7.237 min), HALZ (7.698 min), CLZ (9.081 min), FNZ (9.622 min),LRZ (10.173 min), ALZ (10.800 min), and DZP (15.759 min).


3.3 Sample preparation and extraction

The sample preparation procedure used in this studyinvolved only a single step, i.e., SPE. Four different sorb-ents and two organic solvents (methanol and ACN) in

different compositions have been tested to optimize theSPE protocol by means of the recovery of analytes com-pared to that of unextracted samples. From the resultspresented in Tables 3 and 4, Nexus Varian and 50:50


Figure 3. Chromatogram of (A) blank urine and (B) urine spiked with BDZs (8 ng/lL) under the conditions described in text,where peak sequence is as in Fig. 2.


MeOH/ACN yielded the best recoveries for all analytes.The results were expressed as percentage recovery (i.e.,recovery (%) = (peak area of extracted analyte/peak area

of nonextracted analyte)6100 (%)). Optimization of sam-ple pretreatment involved the result of a clean chromato-gram as well.


Figure 4. Chromatogram of (A) blank saliva and (B) saliva spiked with BDZs (4 ng/lL) under the conditions described in text,where peak sequence is as in Fig. 2.


3.4 Preparation of calibration curves

Calibration curves were prepared for seven concentra-tion levels ranging from 0.2 to 20.0 ng/lL of analytes forstandard and spiked bio-fluids (human plasma, urine,and saliva). The biological samples were analyzed accord-ing to the extraction procedure described in Section 2.The calibration curves were constructed by plotting peakarea ratios of corresponding analytes to the internalstandard against their theoretical concentrations whichwere fitted by a least squares linear regression to theequation: response ratio (y) = slope6concentration(x) + intercept. The regression coefficient (r2) of the cali-bration lines, the day-to-day standard error in the slopesand the intercepts are given in Table 5.

3.5 Method validation

3.5.1 Linearity

Calibration curve is linear over the range 0.2–20 ng/lLstudied in standard solutions for all analytes exceptalprazolam (APZ). For APZ in standard solutions as wellas for all analytes in spiked plasma, urine, and saliva sam-ples linearity holds from 0.3 to 20.0 ng/lL, with coeffi-cients of determination (r2) better than 0.997.

3.5.2 LOD and LOQ

The LOD, defined as the lowest absolute amount of ana-lyte in a sample can be detected but not necessarily quan-tified and the LOQ, the lowest amount of analyte in asample that can be determined with acceptable precisionand accuracy (i.e., f20% (n = 6)) were calculated fromregression equation using the formula: LOD = 3Sxy/a, andthe LOQ: LOQ = 10Sxy/a, where Sxy is the SD of interceptand a is the slope [40].

LOD values were 0.02–0.15, 0.08–0.47, 0.08–0.34 ng/lL, 0.04–0.14 for standard, plasma, urine, and saliva,respectively and corresponding LOQ values were 0.07–0.53, 0.22–1.57, 0.26–1.59 ng/lL and 0.13–0.45 ng/lL.

3.5.3 Accuracy/recovery

The accuracy of the method is defined as the degree ofagreement of test results generated by the method to thetrue value. The analytical recovery (extraction efficiency)was used to assess the accuracy and it was measured byspiking drug-free plasma, urine, and saliva samples withknown concentrations of the standards (QC samples).The QC samples at three different concentration levels(2.0, 4.0, 8.0 ng/lL for standard, plasma, urine, and 0.05,1.0, 2.0 ng/lL for saliva) were assessed for the evaluationof the accuracy of the method. The chromatographicpeak area ratios of analytes extracted from spikedplasma, urine, and saliva at each level of the QC samplesto the area ratio of IS were used to determine the concen-tration using respective calibration equation and it wascompared with the corresponding nominal concentra-tion. The results were expressed as percentage recovery(i.e., recovery (%) = (calculated concentration/nominalconcentration)6100 (%)).

The mean relative recoveries at three concentrationlevels shown in Tables 6–9 for all analytes were 96.3–108.6, 96.0–108.2, 94.3–107.1, 97.0–107.0% in within-day analysis and 96.8–107.7, 94.6–107.6, 93.2–105.8,96.0–108.6% in between-day analysis for standard,plasma, urine, and saliva, respectively.


Table 3. Optimization of SPE sorbent for the isolation of ana-lytes

Analytes Recovery (%)

Lichrolut Varian LC-18 DSC-18

BRZ 92.0 96.7 99.0 91.6HTRZ 98.5 104.9 94.5 89.0HALZ 97.7 105.9 95.0 91.7CLZ 102.4 98.2 102.7 92.4FNZ 94.3 103.9 96.8 91.3LRZ 99.8 97.5 98.4 93.0ALZ 106.1 98.4 108.0 87.6DZP 93.3 105.3 103.9 91.8

Table 4. Optimization of elution solvents for SPE using Nexus Varian sorbent

Eluent composition Recovery (%)

BRZ HTRZ HALZ CLZ FNZ LRZ ALZ DZP

MeOH/ACN = 100:0 98.0 100.1 99.2 102.4 105.8 96.4 98.8 99.8MeOH/ACN = 80:20 96.8 104.0 103.7 105.0 96.8 98.4 95.8 99.3MeOH/ACN = 60:40 93.1 100.8 106.3 104.0 96.1 96.4 94.9 94.4MeOH/ACN = 50:50 96.3 104.1 102.9 102.8 102.7 99.3 96.2 100.9MeOH/ACN = 40:60 80.3 93.4 97.6 94.7 90.6 96.7 85.4 93.9MeOH/ACN = 20:80 60.7 84.3 81.7 86.6 80.7 77.4 78.1 85.2MeOH/ACN = 0:100 20.4 51.7 30.9 90.5 71.0 6.5 88.7 89.6


3.5.4 Assay precision

Precision of a quantitative method is the degree of agree-ment among individual test results when the procedureis applied repeatedly to multiple samplings. The analysiswas carried out in one laboratory by one operator, usingthe same reagents and instruments over a relativelyshort time span. The precision of the method was eval-uated by assaying QC samples at three different concen-trations shown in Tables 6–9. Intra-assay precision(within-day repeatability) was evaluated by analyzing theQC samples (n = 6 for each level) on the same day. Theinter-day precision (between-day reproducibility) is definedas the long-term variability of the measurement process,which here was assessed from the same pooled urine,plasma, and saliva samples as above, by analyzing tripli-cates of each of the QC samples on six successive days

over a 2 wk period. The concentrations of the analytes inthe quality control samples were calculated using cali-bration curves. Within- and between-day precision wereassessed by determining the RSDs (% RSD), calculatedfrom the ratio of SD to the mean of each QC samples ofeach concentration level, and expressed as a percentage.Within- and between-day precision and accuracy data atthe concentrations are shown in Tables 6–9. The within-assay (n = 6) RSD were 0.03–4.7%, 1.3–7.9%, 2.1–6.0%,2.2–5.8% and the between-assay (n = 6) RSD were 0.5–7.0%, 3.3–7.3%, 2.1–7.8%, 2.2–8.1% for standard,plasma, urine, and saliva, respectively.

3.5.5 System suitability

The system suitability was assessed by replicate injec-tions of the sample at a concentration of 2 ng/lL includ-


Table 5. Sensitivity and linearity data of the proposed method

Analytes Regression equation r2 LOD(ng/lL)

LOQ(ng/lL)

Linearity range(ng/lL)

StandardBRZ y = (0.307 l 0.002)x + (0.021 l 0.002) 0.999 0.02 0.07 0.2–20HTRZ y = (0.195 l 0.002)x + (0.024 l 0.006) 0.999 0.09 0.31HALZ y = (0.173 l 0.002)x + (0.032 l 0.004) 0.999 0.07 0.23CLZ y = (0.232 l 0.003)x + (0.014 l 0.008) 0.998 0.10 0.34FNZ y = (0.263 l 0.004)x + (0.029 l 0.014) 0.998 0.15 0.53LRZ y = (0.229 l 0.003)x + (0.020 l 0.003) 0.999 0.03 0.11ALZ y = (0.202 l 0.004)x – (0.021 l 0.01) 0.998 0.15 0.50 0.3–20DZP y = (0.328 l 0.003)x + (0.011 l 0.003) 0.999 0.03 0.091 0.2–20

PlasmaBRZ y = (0.321 l 0.002)x + (0.000 l 0.02) 0.999 0.08 0.22 0.3–20HTRZ y = (0.207 l 0.002)x + (0.047 l 0.02) 0.999 0.29 0.97HALZ y = (0.172 l 0.003)x + (0.101 l 0.03) 0.998 0.47 1.57CLZ y = (0.22 l 0.005)x + (0.136 l 0.015) 0.997 0.21 0.68FNZ y = (0.262 l 0.004)x + (0.064 l 0.04) 0.998 0.46 1.52LRZ y = (0.224 l 0.003)x + (0.060 l 0.03) 0.999 0.35 1.16ALZ y = (0.175 l 0.003)x + (0.039 l 0.03) 0.999 0.43 1.42DZP y = (0.331 l 0.003)x + (0.071 l 0.03) 0.999 0.27 0.90

UrineBRZ y = (0.251 l 0.004)x + (0.139 l 0.04) 0.998 0.16 1.59 0.3–20HTRZ y = (0.207 l 0.002)x – (0.014 l 0.02) 0.999 0.29 0.96HALZ y = (0.183 l 0.002)x – (0.004 l 0.02) 0.999 0.33 1.09CLZ y = (0.2300 l 0.006)x – (0.002 l 0.006) 1.00 0.08 0.26FNZ y = (0.264 l 0.001)x – (0.006 l 0.01) 0.999 0.11 0.38LRZ y = (0.226 l 0.002)x + (0.010 l 0.02) 0.999 0.27 0.88ALZ y = (0.175 l 0.002)x – (0.005 l 0.03) 0.999 0.34 1.14DZP y = (0.318 l 0.001)x + (0.011 l 0.01) 0.999 0.09 0.31

SalivaBRZ y = (0.270 l 0.004)x + (0.040 l 0.02) 0.999 0.11 0.37 0.3–20HTRZ y = (0.200 l 0.001)x + (0.023 l 0.005) 0.999 0.08 0.25HALZ y = (0.177 l 0.001)x + (0.032 l 0.003) 0.999 0.05 0.17CLZ y = (0.233 l 0.002)x + (0.031 l 0.009) 0.999 0.12 0.39FNZ y = (0.254 l 0.004)x + (0.043 l 0.01) 0.999 0.12 0.39LRZ y = (0.229 l 0.004)x + (0.028 l 0.001) 0.999 0.04 0.13ALZ y = (0.175 l 0.008)x + (0.049 l 0.002) 0.999 0.14 0.45DZP y = (0.312 l 0.002)x + (0.042 l 0.009) 0.999 0.09 0.29


ing within- and between-day assessments for standard,spiked plasma, urine, and saliva samples. Precision ofretention times and peak area were examined to evaluatethe system suitability from within-day repeatability(mean value of six measurements, n = 24) and between-day precision (mean value of three measurements duringsix days, n = 72) expressed by percentage RSD, whichrevealed the values of 0.8–1.9% and 0.7–1.7%, respec-tively. Precision (RSD) of retention time of the analytesfrom chromatograms (n = 6) taken at different bufferconcentrations (0.04–0.06 M) was also calculated to eval-uate the system suitability. The results ranged from 0.5to 2.2 as shown in Table 10, indicating excellent suitabil-ity of the system.

3.5.6 Selectivity and specificity

Selectivity of an analytical method is its ability to meas-ure accurately an analyte in the presence of interferencethat may be expected to be present in the sample matri-ces. The specificity was demonstrated, showing that thedetermination of drugs and metabolites was free frominterferences from degradation products. The absence ofany endogenous interfering peak observed in theextracts of bio-fluids overlapping with any analyte or theIS indicates the high specificity of the method which canbe used in therapeutic and routine analyses. Typical

chromatograms of blank samples are presented in Figs.2–4.

3.5.7 Robustness

Robustness is the capacity of the method to remain unaf-fected by small deliberate variations in several chromato-graphic parameters. The optimum HPLC conditions setfor this method have been slightly modified by the smallchanges in flow rate and ammonium acetate concentra-tion to justify the effect (if any) on the results as a meansto evaluate the method's robustness. It was found thatthe percent recoveries of BDZs and metabolites wereexcellent under most conditions, and remained unaf-fected by small deliberate changes of flow rate as shownin Table 3. Inter-method verification has also been eval-uated as an evidence of robustness test, when a previ-ously developed method was applied to measure theaccuracy of six studied parent drugs.

The effect of varying ammonium acetate concentra-tions from 0.04 to 0.06 M to the peak area, retentiontime, resolution factor was not significant and columnperformance was not affected.

The study of variation of the flow rate from 1.0 to1.1 mL/min showed that the peak area decreased atincreased flow rate for all the compounds. This is due tothe fact that at high flow rates the compounds do nothave time to penetrate the pores of particles and to be


Table 6. Within- and between-day accuracy and precision data in standards

Analytes Added(ng/lL)

Within-day (n = 6) Between-day (n = 6)

Obtained(ng/lL)

R (%) RSD(%) Obtained(ng/lL)

R (%) RSD (%)

BRZ 2.0 2.17 l 0.03 108.6 3.9 1.99 l 0.01 99.7 2.04.0 4.05 l 0.04 101.3 3.1 4.01 l 0.05 100.2 4.28.0 7.74 l 0.05 96.8 2.3 7.87 l 0.01 98.4 0.5

HTZL 2.0 2.03 l 0.01 101.7 2.7 2.03 l 0.02 101.4 4.54.0 4.27 l 0.02 106.8 2.3 4.22 l 0.03 105. 6 3.78.0 8.16 l 0.06 102.0 3.6 7.98 l 0.02 99.7 1.3

HAPZ 2.0 2.08 l 0.01 103.9 3.4 2.05 l 0.02 102.4 5.34.0 4.31 l 0.02 107.8 2. 3 4.31 l 0.04 107.7 4.88.0 8.22 l 0.06 102.8 4.1 7.89 l 0.08 98.6 5.8

CLZ 2.0 2.01 l 0.02 100.5 4.7 2.04 l 0.03 101.9 7.14.0 4.37 l 0.02 109.3 1.8 4.25 l 0.05 106.3 5.48.0 8.30 l 0.09 103.8 4.6 8.11 l 0.03 101.4 1.6

FNZ 2.0 2.02 l 0.02 100.8 3.7 1.98 l 0.02 99.0 4.24.0 4.25 l 0.02 105.9 1.5 4.19 l 0.05 104.7 4.48.0 8.04 l 0.09 100.6 3.9 7.91 l 0.05 98.9 2.2

LRZ 2.0 2.02 l 0.01 101.1 2.8 2.02 l 0.03 101.1 5.24.0 4.19 l 0.01 104.8 0.1 4.12 l 0.05 102.9 4.78.0 7.87 l 0.05 98.4 2.9 7.83 l 0.02 97.9 1.0

APZ 2.0 1.96 l 0.01 98.0 3.6 1.95 l 0.02 97.7 5.84.0 4.04 l 0.02 101.1 0.1 3.93 l 0.04 98.2 5.08.0 7.71 l 0.02 96.3 1.4 7.74 l 0.04 96.8 2.4

DZP 2.0 2.03 l 0.02 101.3 3.4 2.14 l 0.02 107.2 2.94.0 4.30 l 0.02 107.6 1.7 4.18 l 0.06 104.5 4.18.0 8.11 l 0.11 101.4 4.0 7.77 l 0.08 97.2 3.2


retained by the hydrophobic inner surface of the par-ticles before being eluted by the analytical mobile phase.Also at high flow rate the retention time of each peakwas reduced with subsequent change in the separationand resolution factor without affecting their good sep-aration. However, 1.1 mL/min seems to be a good com-promise when considering the chromatographic systemand total retention time and precision of peak area.

3.5.8 Stability study

3.5.8.1 Standard solution

The stability of standard solutions was tested by the pro-posed HPLC method over a period of 90 days. The freshlyprepared solutions at room temperature, and the90 days stored samples in a refrigerator at 48C, were ana-lyzed. Chromatograms and recoveries were compared.No degradation products were present and the drugs arestable at 48C for at least 90 days, indicating the possibil-ity of using all studied drugs over a period of 90 daysstored at refrigerator without degradation. Degradationwas decided by using the –10% criterion.

3.5.8.2 Spiked solution

Freeze –thaw cycle of spiked samples: In case of biologi-cal samples, plasma, urine, and saliva samples spikedwith 2 ng/lL of analytes were stored deep-frozen at

–258C. Each sample was analyzed once daily for sevensuccessive days after freeze–thaw cycle for the investiga-tion of stability when spiked samples were allowed tothaw at room temperature. Recovery of analytes for thestored samples was calculated and compared to that offreshly prepared samples. The results show that up to thethird cycle all analytes were almost stable and up to fifthcycle, recovery was decreased by less than 10%. Resultshave been presented graphically in Fig. 5.

4 Discussion

Although GC methods are more sensitive than HPLCmethods for measuring BDZs and metabolites in biologi-cal interest, they involve lengthy sample clean-up proce-dures, and require derivatization steps to increase thevolatility of BDZs, which are thermally unstable underGC conditions. In addition, they require a suitable IS toavoid the thermal-molecular rearrangement that occurson the column which may lead to poor reproducibility ofthe assay.

The proposed chromatographic method has theadvantage to detect and to quantitate the whole spec-trum of effective compounds and metabolites in a singlerun under optimized conditions. HPLC offers the advant-age to separate and quantitate the compounds without


Table 7. Within- and between-day accuracy and precision data in plasma samples

Analytes Spiked (ng/lL) Within-day (n = 6) Between-day (n = 6)

Obtained(ng/lL)

R (%) RSD (%) Obtained(ng/lL)

R (%) RSD (%)

BRZ 2.0 2.12 l 0.03 105.9 3.9 2.08 l 0.04 103.8 5.34.0 3.88 l 0.02 97.1 1.4 3.98 l 0.05 99.6 4.28.0 7.87 l 0.09 98.4 3.4 7.91 l 0.15 98.8 5.8

HTZL 2.0 1.99 l 0.01 99.3 1.3 2.14 l 0.02 107.2 4.14.0 3.91 l 0.04 97.7 4.6 3.92 l 0.04 97.9 5.38.0 7.68 l 0.06 96.0 3.6 7.57 l 0.11 94.6 6.8

HAPZ 2.0 2.08 l 0.04 103.9 7.9 2.11 l 0.02 105.7 3.34.0 4.26 l 0.02 106.5 1.9 3.94 l 0.03 98.6 4.28.0 7.96 l 0.05 99.5 3.5 7.86 l 0.10 98.3 7.2

CLZ 2.0 2.16 l 0.02 108.2 4.0 2.15 l 0.03 107.6 5.04.0 3.99 l 0.8 99.7 7.4 4.02 l 0.05 100.6 4.68.0 8.21 l 0.07 102.6 3.4 8.10 l 0.13 101.2 6.7

FNZ 2.0 2.18 l 0.02 109.0 3.6 2.04 l 0.04 102.1 7.14.0 4.12 l 0.6 103.0 4.9 4.11 l 0.06 102.9 5.58.0 8.02 l 0.07 100.3 3.3 7.78 l 0.15 97.2 7.2

LRZ 2.0 2.03 l 0.01 101.6 2.5 2.06 l 0.04 103.0 6.74.0 4.13 l 0.06 107.3 6.3 4.19 l 0.05 104.7 5.48.0 8.00 l 0.06 100.0 3.3 7.94 l 0.13 99.3 7.0

APZ 2.0 2.14 l 0.01 107.2 2.5 2.02 l 0.01 101.1 3.44.0 4.24 l 0.05 106.1 6.1 4.22 l 0.03 105.6 3.98.0 8.24 l 0.04 103.0 2.5 8.19 l 0.11 102.4 7.3

DZP 2.0 2.03 l 0.02 101.4 3.1 2.10 l 0.05 104.9 6.84.0 4.02 l 0.06 100.5 4.4 4.01 l 0.07 100.4 4. 98.0 7.86 l 0.09 98.2 3.5 7.65 l 0.16 95.6 6.2


derivatization and at low temperature without the riskof decomposition. It is known that the combination ofHPLC with DAD is considered as a highly effective screen-ing method. The use of the DAD also gives the advantageof identifying the analytes both by retention time andUV spectrum. Furthermore, most of the previouslyreported techniques with higher sensitivities required acomplex extraction procedure and the use of large sam-ple volumes (F1 mL), which is not practical for pharma-cokinetic studies, where taking large volumes of blood isethically unacceptable.

Usually two hydrolysis procedures, acidic or enzy-matic, can be used on the conjugates prior to extractionfor urine samples. But BDZs are converted to benzophe-nones by acid hydrolysis and this may give difficulties toidentify the parent compounds. BDZs are known to bindthe protein albumin but mainly on a-glycoprotein. How-ever, organic solvents miscible with water, e.g. ACN, candecrease the solubility of proteins, precipitate themfrom aqueous solutions, and render them removable bycentrifugation.


Table 8. Within- and between-day accuracy and precision data in urine samples


Obtained(ng/lL)


R (%) RSD (%)

BRZ 2.0 1.96 l 0.03 98.1 4.4 2.02 l 0.04 101.2 7.44.0 3.91 l 0.04 97.8 4.1 4.16 l 0.06 103.9 5.28.0 8.57 l 0.08 107.1 3.7 8.46 l 0.13 105.8 5.4

HTZL 2.0 1.96 l 0.01 97.9 2.7 1.91 l 0.02 95.6 5.74.0 3.77 l 0.02 94.3 2.7 3.91 l 0.04 97.9 4.68.0 7.68 l 0.05 96.0 3.6 7.86 l 0.14 98.3 8.7

HAPZ 2.0 1.95 l 0.01 97.6 2.1 1.93 l 0.02 96.5 4.44.0 3.81 l 0.02 95.2 3.6 3.97 l 0.04 99.2 5.58.0 7.73 l 0.05 96.6 3.5 7.93 l 0.09 99.1 6.4

CLZ 2.0 1.94 l 0.02 97.2 4.0 1.91 l 0.02 95.6 4.34.0 3.78 l 0.03 94.6 3.8 4.06 l 0.04 101.5 4.48.0 7.67 l 0.06 95.9 3.7 8.37 l 0.11 104.7 5.7

FNZ 2.0 2.00 l 0.03 100.0 5.8 1.91 l 0.01 95.7 2.14.0 3.83 l 0.03 95.9 3.3 3.93 l 0.04 98.4 3.88.0 7.63 l 0.12 95.3 6.0 7.97 l 0.13 99.6 6.0

LRZ 2.0 1.97 l 0.01 98.6 3.4 1.89 l 0.01 94.5 2.14.0 3.84 l 0.03 95.9 4.1 3.92 l 0.04 98.0 5.08.0 7.71 l 0.10 96.4 6.0 8.17 l 0.14 102.1 7.8

APZ 2.0 1.97 l 0.02 98.7 5.2 1.86 l 0.02 93.2 5.14.0 3.92 l 0.02 98.1 2.4 4.14 l 0.05 103.5 7.68.0 7.72 l 0.05 96.5 3.8 8.32 l 0.12 104.0 8.2

DZP 2.0 1.96 l 0.02 98.2 3.6 1.94 l 0.02 97.3 3.34.0 3.94 l 0.05 98.4 3.8 3.91 l 0.06 97.9 5.18.0 7.71 l 0.09 96.4 3.9 7.87 l 0.10 98.3 4.1

Figure 5. Stability study data of freeze–thaw cycles.


In SPE method, no interferences were found using the50:50 mixture of methanol/ACN as eluting solvent to geta fair chromatogram with expected recoveries of bothBDZs and metabolites when hydrophilic impurities werecompletely removed giving clear extract by washingwith 1 mL of water before elution.

Colchicine, having the good resolution and separationfactors between adjacent peaks was used as internalstandard. Freshly prepared quality controlled solutionswere used for analysis because solutions containing ISafter long time storage showed interfering peaks prob-

ably due to the photodecomposition or chemical interac-tion forming hydrogen bond with the analytes. Alterna-tively, reconstitution of eluted drugs after dryness on N2

was recommended by the methanolic solution of IS atcertain concentration to avoid the problem.

Analytical methods for BDZs in biological fluids areproblematic, due to the high sensitivity required todetect them as their low concentration in biofluids. Inaddition it is challenging to separate and isolate themetabolites simultaneously due to their high polarity ascompared to the parent drugs. There are so many active


Table 9. Within- and between-day accuracy and precision data in saliva samples


Obtained(ng/lL)


R (%) RSD (%)

BRZ 0.5 0.53 l 0.004 105.3 2.4 0.53 l 0.01 105.4 2.81.0 1.01 l 0.01 101.2 4.2 1.06 l 0.03 106.5 8.02.0 2.11 l 0.02 105.4 3.8 2.08 l 0.03 104.2 5.6

HTZL 0.5 0.53 l 0.005 107.0 3.8 0.50 l 0.01 100.0 8.11.0 1.02 l 0.008 101.6 3.7 0.96 l 0.01 96.4 6.22.0 2.00 l 0.01 100.0 2.5 2.15 l 0.01 107.5 2.2

HAPZ 0.5 0.49 l 0.003 97.2 3.0 0.50 l 0.01 100.0 5.51.0 0.97 l 0.01 97.0 5.5 0.99 l 0.02 99.0 7.42.0 1.98 l 0.008 99.0 2.2 2.14 l 0.01 107.0 2.2

CLZ 0.5 0.51 l 0.005 101.2 3.6 0.49 l 0.01 98.0 6.51.0 1.03 l 0.02 103.3 5.7 0.97 l 0.01 97.0 4.92.0 2.01 l 0.02 100.5 3.4 2.17 l 0.02 108.6 3.5

FNZ 0.5 0.49 l 0.007 98.8 4.4 0.48 l 0.01 96.0 4.71.0 0.99 l 0.02 99.0 5.8 0.96 l 0.01 96.0 3.22.0 1.98 l 0.02 98.8 3.1 2.11 l 0.03 105.3 5.4

LRZ 0.5 0.52 l 0.007 103.3 5.3 0.49 l 0.01 98.0 6.41.0 1.03 l 0.01 103.0 5.0 1.02 l 0.02 102.4 7.52.0 2.04 l 0.02 101.9 3.9 2.09 l 0.02 104.6 3.4

APZ 0.5 0.52 l 0.005 103.1 3.3 0.50 l 0.01 100.0 6.81.0 1.01 l 0.01 101.3 5.4 0.98 l 0.01 98.0 6.52.0 2.00 l 0.02 100.0 5.5 2.17 l 0.02 108.3 3.7

DZP 0.5 0.49 l 0.008 98.4 4.3 0.49 l 0.01 98.0 7.61.0 1.01 l 0.01 101.2 3.2 1.07 l 0.02 106.7 6.12.0 1.96 l 0.02 98.0 3.0 2.12 l 0.05 105.8 7.7

Table 10. System suitability and method robustness data

Analytes RSD (%) Buffer effects (CH3COONH4) Inter-methodcoefficient

Flow rate1.0 mL/min

Flow rate1.1 mL/min

0.04 M 0.05 M 0.06 M RSD ofRt (%)

of determina-tion

Area Rt Area Rt R (%) Rt R (%) % R

BRZ 4.48 0.43 1.34 1.03 96.8 5.47 l 0.06 99.7 97.7 0.5 0.999HTZL 3.47 0.56 1.34 1.44 98.6 7.17 l 0.11 101.4 101.4 0.8 –HAPZ 3.29 0.61 1.36 1.43 99.7 7.66 l 0.11 102.4 104.0 0.7 –CLZ 4.25 0.39 1.21 1.46 101.5 9.00 l 0.13 99.0 99.2 2.2 0.999FNZ 3.71 0.56 1.30 1.56 99.4 9.55 l 0.15 99.0 96.3 1.7 1.000LRZ 1.18 0.63 1.10 1.89 96.7 10.17 l 0.19 101.1 99.4 0.9 0.999APZ 1.31 0.41 0.70 1.89 97.2 10.77 l 0.21 97.7 98.4 0.8 0.999DZP 3.98 0.16 1.66 0.79 105.5 15.77 l 0.12 107.2 106.1 1.1 0.999


metabolites of studied drugs but either they are not com-mercially available, or at a high cost. Two a-hydroxymetabolites of alprozolam and triazolam were success-fully separated and determined.

The method provides satisfactory sensitivity and preci-sion within considerable analytical range. LOQs forplasma are supposed to be higher than those in urineand saliva. This was expected due to the complex chemi-cal composition from interfering drug release.

5 Conclusions

The HPLC analytical method for the determination ofBDZs in biofluids presented herein meets the criteria forroutine therapeutic drug monitoring or pharmacoki-netic studies. The advantage of the method over previ-ously reported methods is the rapidity, simplicity (easeof sample preparation), high sensitivity, and high selec-tivity. The LOD and LOQ achieved make the assay appro-priate for the measurement of therapeutic concentra-tions.

Saliva should not be seen as a specimen that replacesthe use of other specimens as drugs concentration is toolow. Urine should still be seen as the specimen of choicefor BDZs if evidence of prior exposure to drugs of abuse issought (e.g., routine workplace screening). If evidence ofrecent use (or abstinence) of drugs is sought then salivasamples are preferred specimens.

The authors declared no conflict of interest.

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Validation of SPE-HPLC determination of 1,4-benzodiazepines and metabolites in blood plasma, urine,...

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