Current Pharmaceutical Analysis, 2012, 8, 00-00 1

1573-4129/12 $58.00+.00 © 2012 Bentham Science Publishers

Development and Validation of UHPLC/ESI-Q-TOF-MS Method for Terbutaline Estimations in Experimental Rodents: Stability Effects and Plasma Pharmacokinetics

Md. Faiyazuddin1,2,

*, Niyaz Ahmad1, Zeenat Iqbal

1, Sushma Talegaonkar

1, Aseem Bhatnagar

3,

Roop Krishen Khar1, Farhan Jalees Ahmad

1

1Department of Pharmaceutics, Faculty of Pharmacy, Hamdard University, New Delhi-110062, India;

2Department of

Pharmaceutics, Faculty of Pharmacy, Integral University, Lucknow, Uttar Pradesh, India; 3Department of Nuclear

Medicine, Institute of Nuclear Medicine & Allied Sciences (INMAS), Brig. S. K. Mazumdar Road, Delhi-110054, India

Abstract: An ultra high performance liquid chromatography-electrospray ionization-tandem mass spectrometric method

(UHPLC/ESI-Q-TOF-MS) for the analysis of terbutaline (TB) in Wistar rat plasma has been developed and validated. The

chromatographic separation was achieved on a Waters ACQUITY UPLCTM

BEH C18 (100.0 mm 2.1 mm; 1.7 m) col-

umn using isocratic mobile phase, consisting of 2 mM ammonium acetate and acetonitrile (90: 10; v/v), at a flow rate of

0.25 mL min-1

. The transitions occurred at m/z 226.19 152.12 for TB, and m/z 260.34 183.11 for the internal standard.

The recovery of the analytes from Wistar rat plasma was optimized using liquid- liquid extraction technique (LLE) in

ethyl acetate. The total run time was 3.0 min and the elution of TB occurred at 1.85±0.05 min. The linear dynamic range

was established over the concentration range 1–1000 ng mL-1

(r2; 0.9938±0.0005) for TB. The intra-assay and inter-assay

accuracy in terms of % CV was in between 1.8–3.5. The lower limit of quantitation (LLOQ) for TB was 1.0 ng mL-1

.

Analytes were stable under various conditions (in autosampler, during freeze–thaw, at room temperature, and under deep-

freeze conditions). The developed method was successfully applied for pharmacokinetic profiling in rodents.

Keywords: Asthma, Bronchodilator, Ex-vivo Stability, In-vitro assay, Matrix effect, Pharmacokinetics, Terbutaline; UHPLC/ESI-Q-TOF-MS, Validation.

1. INTRODUCTION

Terbutaline (TB), an amphiphilic -[(tert-butylamino) methyl]-3,5-dihydroxy- benzyl alcohol is a synthetic 2-adrenoceptor ( 2AR) stimulant that is widely used to treat bronchial asthma (BA), chronic obstructive pulmonary dis-eases (COPD) and related disorders [1, 2]. There has been a plethora of analytical methods reported for the quantitative estimation of TB in bulk, pharmaceutical formulations and biomatrices mainly based on high performance-liquid chro-matography (HPLC) [3-11], liquid chromatography-tandem mass spectrometry (LC/MS/MS) [12, 13] and gas chroma-tography-mass spectrometry (GC-MS) [14, 15]. These pub-lished methods either involve cumbersome sample prepara-tion steps or are insufficiently sensitive or precise to perform TB analysis. A validated HPLC method for the determina-tion of TB and its possible degradation products has been reported in literature [16], utilizing Hypersil 100 C18, 150 4.6 mm (5 m) column, found inadequate sensitivity of the method due to high limit of quantitation (LOQ; 0.11 g mL

-1) and limit of detection (LOD; 0.37 g mL

-1) values. In

our previous study, a stability-indicating high performance-thin layer chromatography (HPTLC) has been developed and validated for the direct estimation of TB and its potential degradants in bulk and submicronized dry powder inhalers

*Address correspondence to this author at the Department of Pharmaceutics, Faculty of Pharmacy, Hamdard University, New Delhi-110062, India; Tel: +91-7668963486; E-mail: md.faiyazuddin2008@gmail.com

[17]. The low value of LOQ (28.35 ng spot–1

) and LOD (9.41 ng spot

–1) indicated adequate assay sensitivity of our

reported method. Recently, a chiral liquid chromatography–tandem mass spectrometric method (LC–MS/MS) for the simultaneous analysis of bambuterol and its active metabo-lite terbutaline in Wistar rat plasma has been reported [18]. In this method, the chromatographic separation was achieved on a teicoplanin-containing Chirobiotic T chiral (250 mm 4.6 mm; 5 m) column at a flow-rate of 0.4 mL min

-1,

however, and has the disadvantage of being time-consuming because of longer retention time (RT >15 min) and lengthy processing steps. All these studies have further emphasized the need to perform rapid and sensitive quality-control analysis of TB with shorter retention time and improved ef-fects. An approach to chiral separation and analysis of TB enantiomers by chiral complexation and electrospray high-field asymmetric waveform ion mobility spectrometry cou-pled to mass spectrometry (ESI-FAIMS-MS) has been also reported in literature [19]. The limit of detection is 0.10% ( )-terbutaline in a sample of (+)-terbutaline, which is the limiting factor of the method. In a recent study, TB sulfocon-jugates were examined in urinary samples by LC/ESI-MS/MS on Orbitrap mass spectrometer [20]. However, this method was not sensitive enough to detect TB in urine on single dose administration. Therefore a hyphenated chroma-tographical technique with advanced feature is desired to compensate the aforesaid loopholes. However, UHPLC is a novel chromatographic technique utilizing high linear veloci-ties, which is based on concept using columns with smaller

2 Current Pharmaceutical Analysis, 2012, Vol. 8, No. 2 Faiyazuddin et al.

packing (1.7-1.8 m porous particles) and operated under high pressure (up to 15,000 psi). This is an extremely power-ful approach which dramatically improves peak resolution, sensitivity and speed of analysis [18, 21]. In addition, time-of-flight mass spectrometry (Q-TOF-MS) allows the genera-tion of mass information with higher accuracy and precision. UHPLC is specially designed to resist higher back-pressures, with the advantages of fast injection cycles, low injection volumes, negligible carryover and temperature control (4–40 ºC), which collectively contributes to speedy and sensitive analysis [22]. Furthermore, acquity UHPLC columns contain hybrid X-Terra sorbent, which utilizes bridged ethylsiloxane/ silica hybrid (BEH) structure, ensures the column stability under the high pressure and wide pH range (1–12) [22]. In addition to UHPLC, the use of orthogonal quadrupole time-of-flight mass spectrometry (Q-TOF-MS), with low and high collision-energy full scans acquisition simultaneously per-formed, offers more possibilities in screening and identifica-tion, resulting in valuable fragmentation information [17, 22, 23]. Consequently UHPLC/ESI-Q-TOF-MS has been proved to be a powerful hyphenated technique for bioanalytical in-vestigation [23, 24].

In this paper, for the first time, a validated assay of TB by UHPLC/ESI-Q-TOF-MS was developed and successfully implicated for bioanalytical investigations. This method ex-hibited excellent performance with respect to high sensitivity and lesser retention time.

2. MATERIAL AND METHODS

2.1. Chemicals

Terbutaline (Assigned purity >99.5%; M.P. 248 ºC) was purchased from Netco Ltd., India. Propranolol hydrochloride as internal standard was a gift from Jubilant Clinsys Clinical Research Limited, Noida, India. HPLC-MS grade acetoni-trile (Assigned purity: 99.9%) was purchased from Sigma-Aldrich, Germany. Solvents used for UHPLC were of gradi-ent grade purity and bought from Merck (Darmstadt, Ger-many). MS grade ammonium acetate and ammonium for-mate were obtained from Fluka analytical, Sigma-Aldrich, Netherland. Formic acid (Assigned purity >98%) was com-mercially obtained from Fluka analytical, Germany. Deion-ized water was purified using a Milli-Q water purification system (Millipore, Bedfrod, MA, USA). Blank plasma from Wistar rats was provided by Yash laboratories, Mumbai, India.

2.2. UHPLC Conditions

UHPLC was performed with a Waters ACQUITY UPLC

TM system (Waters Corp., MA, USA) equipped with a

binary solvent delivery system, an auto-sampler, column manager and a tunable MS detector (Synapt; Waters, Man-chester, UK). Chromatographic separation was performed on a Waters ACQUITY UPLC

TM BEH C18 (100.0 mm 2.1

mm; 1.7 m) column. The mobile phase for UHPLC analysis consisted of acetonitrile–2 mM ammonium acetate (1: 9; v/v), which was degassed. For isocratic elution, the flow rate of the mobile phase was kept at 0.25 mL min

-1 and 10 L of

sample solution was injected in each run. The total chroma-tographic run time was 3.0 min.

2.3. Q-TOF-MS Conditions

Mass spectrometry was performed on a Waters Q-TOF Premier (Micromass MS Technologies, Manchester, UK) mass spectrometer. The Q-TOF Premier

TM was operated in V

mode with resolution over 8500 mass with 1.0 min scan time, and 0.02 s inter-scan delay. Argon was employed as the collision gas at a pressure of 5.3 10

-5 Torr. Quantitation was

performed using Synapt Mass Spectrometery (Synapt MS) of the transitions of m/z 226.19 152.12 for TB and m/z 260.34 183.11 for IS (Propranolol) respectively with a scan time of 1.0 min scan time, and 0.02 s inter-scan per transi-tion. The optimum values for compound-dependent parame-ters like trap collision energy (Trap CE) and transfer colli-sion energy (Tran CE) were set to 13.2 and 11.9 V, respec-tively for fragmentation information. The accurate mass and composition for the precursor ions and for the fragment ions were calculated using the MassLynx V 4.1 software.

2.4. Quality Control (QC) Sample and Standard Prepa-

ration

The standard stock solution of 100 g mL-1

of TB was prepared by dissolving requisite amount in water sonicated at 44 kHz/250W for 20 min. Calibration curve (CC) standards consisting of a set of ten non-zero concentrations (A-J) were prepared by 5% aqueous analytes spiking in blank rat plasma (50 L aqueous aliquots to 950 L blank rat plasma) yield-ing concentration range from 1–1000 ng mL

-1 for TB. The

final concentrations for each analyte were prepared to be 1, 2, 5, 10, 20, 50, 100, 250, 500, 750 and 1000 ng mL

-1. QC

samples were prepared independently at three levels; 800 ng mL

-1 (HQC, high quality control), 402 ng mL

-1 (MQC, mid-

dle quality control) and 2 ng mL-1

(LQC, low quality con-trol). A 100 ng mL

-1, internal standard working solution was

prepared by diluting the stock solution in methanol-water (50: 50 v/v). All the solutions were stored at 2–8 ºC until use.

2.5. Sample Preparation Protocol

All the solutions (CC standards, QC samples and un-

known plasma samples) were freshly prepared before carry-

ing out the experiments. The 200 L aliquot of each samples

were taken into glass tube, 50 L of IS (100 ng mL-1

) was

added in each sample, and further 500 L of formic acid (5%

w/v) was incorporated to mixture and vortexed at 300 rpm

for 5 min). Finally, 10 L of plasma was extracted with 1

mL of ethyl acetate, and the reaction mixture was evaporated

to 900 L. 50 L of reconstitution solution (acetonitrile:

ammonium acetate buffer; 10: 90, v/v) was then added and

vortexing for 10 s at 300 rpm, the solution was transferred

into the clean autosampler vials and 10 L was injected into UHPLC/MS system for analysis.

2.6. Bioanalytical Method Validation

The method validation of TB in rodent plasma was per-formed according to USFDA guidelines [21]. The linearity of the method was determined by analysis of five standard plots containing ten non-zero concentrations. Peak area ra-tios of analyte/ IS were utilized for the construction of cali-bration curves, using weighted (1/x

2) linear least squares

UHPLC/ESI-Q-TOF-MS Assay of Terbutaline Current Pharmaceutical Analysis, 2012, Vol. 8, No. 2 3

regression of the plasma concentrations and the measured peak area ratios. The lower limit of quantification (LLOQ) is the lowest concentration of the calibration curve, which could be measured with acceptable accuracy and precision. The LLOQ was determined based on the signal-to noise ratio of 10:1. The extraction efficiency (recovery) of TB was per-formed at LQC, MQC and HQC levels. It was evaluated by comparing the mean area response of six replicates of ex-tracted samples (spiked before extraction) to that of extracted drug free plasma samples (spiked after extraction) at each QC levels. The recovery of IS was similarly estimated. For determining the intra-day accuracy and precision, replicate analysis of plasma samples of TB was performed on the same day. The run consisted of a calibration curve and six replicates of LQC, MQC and HQC samples. The inter-day accuracy and precision were assessed by analysis of six pre-cision and accuracy batches on three consecutive validation days. However, for evaluating the ruggedness of the method, one batch of precision and accuracy was run using a different column (same type) by a different analyst employing the same or another instrument. The six replicates were run for LQC, MQC and HQC samples.

2.7. Matrix Effect

To study the effect of matrix on analyte quantification 6 samples were prepared from 6 different batches of plasma at LQC and HQC levels and checked for the % accuracy and precision (%CV) in both the QC samples. This was assessed by comparing the back calculated value from the QC’s nominal concentration. After specified storage conditions, samples were processed and analyzed. The matrix effect was investigated by postextraction spike method. Peak area (A) of the analyte in spiked blank plasma with a known concen-tration (MQC) was compared with the corresponding peak area (B) obtained by direct injection of standard in the mo-bile phase. The ratio (A/B 100) is defined as the matrix ef-fect.

2.8. Ex Vivo Stability

The stability of TB in rodent plasma was evaluated by analyzing six replicates of plasma samples at the concentra-tions of 2 ng mL

-1 (LQC) and 800 ng mL

-1 (HQC) which

were exposed to different conditions (time and temperature). Percentage stability was determined as; % Stability= mean corrected response of stability stock/ mean response of com-parison stock 100.

2.8.1. Long Term Stability

The long-term stability was assessed after storage of the standard spiked plasma samples at deep freeze ( 80 C) for one month. Six replicates of LQC and HQC were used for analysis.

2.8.2. Freeze Thaw Stability

The freeze/thaw stability in plasma was evaluated for three consecutive freeze–thaw cycles from 20 ºC to room temperature (+25 ºC). Six replicates of LQC and HQC were analyzed after undergoing three freeze-thaw cycles.

2.8.3. Bench Top Stability

Bench top stability was determined for 24 h storage in optimized conditions, using six sets each of LQC and HQC.

The QC samples were quantified against the freshly spiked calibration curve standards

2.8.4. Post Processing Stability

Short-term stability was determined after the exposure (of processed samples) at 10 ºC for 24 h in autosampler us-ing six sets each of LQC and HQC. After specified storage conditions, samples were processed and analyzed. The ana-lytes are considered to be stable when the precisions are be-low 15% and the accuracies are in the range of 85–115% respectively for both levels [12].

2.9. Pharmacokinetic Investigation In Vivo

2.9.1. Experimental Animal

Wistar rats (n=6; 200–250 g, 8–10 weeks old) were pro-vided from Experimental Central Animal House, Hamdard University and kept in an environmentally controlled room (Temperature: 25±2 °C, humidity: 60±5%, 12 h dark–light cycle) for at least 1 week before the experiments. Animals were fed on standard pelleted diet (Ashirwad Industries, Chandigarh, India) and water was provided ad libitum. The rats were fasted overnight before the day of the experiment.

2.9.2. Experimental Design Protocol

The animal protocol used in this study was approved by Hamdard University’s Institutional Animal Ethics Commit-tee. Rats were fasted for 12 h with free access to water prior to the pharmacokinetic investigation. The bioanalytical method was implicated for quantitative estimation of TB in Wistar rat’s plasma after a single oral dose (5mg kg

-1 dis-

solved in distilled water, given by oral feeding tube). Using venous catheter, 1 mL of blood samples were withdrawn and procured in pre-heparinized glass tubes at different time in-tervals (0, 0.083, 0.166, 0.25, 0.5, 1, 2, 3, 4, 6, 8, 12, and 16 h). Further, blood samples were centrifuged (2500 g; 10 min; 20 ºC) to separate plasma fractions. The collected plasma samples were preserved for investigation at 80 C until analysis. Concentration–time curves were established for each analyte and used for the determination of pharma-cokinetic parameters such as peak plasma concentration (Cmax), peak time (Tmax), extent of absorption (AUC) and half-life (T0.5), by a non-compartmental analysis using PK Solutions Version 2.0; Summit Research Services, USA.

3. RESULTS AND DISCUSSION

3.1. UHPLC/ESI-Q-TOF-MS/MS Analysis

TB is a low molecular weight compound (MW; 226),

containing a secondary amine in its structure. Due to the

presence of basic nitrogen in the molecule, TB exhibited favorable sensitivity in positive ion mode detection. Opti-

mum chromatographic separation of TB, was achieved by

acetonitrile: ammonium acetate buffer (10: 90; v/v), with a flow rate of 0.25 mL min

-1. Baseline separation of both TB

and IS was obtained within runtime of 3 mins, without any

interference. Methanol, acetone and isopropyl alcohol have been also tried for mobile phase selection but they didn’t

afford chromatographic separation. Although various buffer

systems were studied but the fine peak with better signal response was observed for ammonium acetate buffer, due to

4 Current Pharmaceutical Analysis, 2012, Vol. 8, No. 2 Faiyazuddin et al.

its volatile and compatible nature. The MS full scan spectra for TBS showed protonated precursor [M+H]

+ ions at m/z

226.19 and product ion mass spectra at m/z 152.12 respec-

tively (Figs. 1A and 1C). During direct infusion, the mass spectra of IS showed precursor ion peaks at m/z 260.34 as

[M+H]+ ions and most abundant product ions at m/z 183.11

(Figs. 1B and 1D). The optimum collision energies em-ployed were 13.0 and 16.3 eV for TB and IS, respectively.

Quantification was done on the basis of main product ions.

Identical capillary voltage of 3.2 KV was used for monitor-ing the precursor ions.

The most widely employed biological sample preparation

methodologies currently are liquid–liquid extraction (LLE), protein precipitation (PPT), and solid-phase extraction

(SPE). In the early stage of method development, a PPT

method was employed to separate TB from plasma samples, but strong ion suppression from the endogenous substances

in plasma occurred (at 1 min). Although it could be de-

creased by chromatographic separation, the run time would be sacrificed. Finally, liquid–liquid extraction (LLE) proce-

dures were used to prepare TB plasma samples in our study.

To obtain optimum recovery, four organic extraction sol-vents were evaluated including ethyl ether, ethyl acetate,

ethyl ether–dichloromethane (2: 1; v/v) and n-hexane–

dichloromethane (2: 1; v/v). It was found that ethyl acetate alone could yield the highest recovery (>80%) for TB and

IS. A HPLC method reported for TB estimation by Daragh-

meh et al. (2002) used Hypersil 100 C18 column which showed inadequate sensitivity and elaborated retention time

[16]. For a LC–MS/MS method recently reported by Luo et

al. (2010) had longer retention time (RT >15 min) and lengthy processing steps using teicoplanin-containing Chiro-

biotic T chiral column [18]. However, we used Waters AC-

QUITY UPLCTM

BEH C18 column with 1.7 m particle size which helped in separation and elution of both analytes, TB

and IS at 1.85 (±0.05) and 2.40 (±0.08) min, respectively (Fig. 2). The elution of TB (100 ng mL

-1) occurred at 1.87

min (Fig. 2A) and IS (100 ng mL-1

) at 2.40 min (Fig. 2B).

Chromatogram of blank plasma (extracted and reconstituted) is shown Fig. (2C); however TB spiked plasma sample (1 ng

mL-1

) retained at 1.90 (Fig. 2D).

3.2. Bioanalytical Method Validation

3.2.1. Linearity

The calibration curves of TB were linear over the con-centration range of 1–1000 ng mL

-1. The least squares re-

gression analysis gave the linear equation y = 0.0749 0.0038X where y is the peak area ratio of the analyte to the IS and x is the concentration of the analyte. The accu-racy and precision (%CV) observed for the calibration curve standards ranged from 93.34–102.06 % and 1.01–4.07, re-spectively. The LLOQ achieved was 1.0 ng mL

-1 for TB.

3.2.2. Recovery, Accuracy and Precision

No endogenous peak was observed at the retention time of the analyte and IS both for any of the batches. Representa-tive chromatograms (Fig. 2C) of extracted blank plasma for-tified with IS and blank plasma fortified with TB demon-strates the selectivity of the method. The mean recovery (n = 6) for TB at HQC, MQC and LQC levels was 91.59, 85.6 and 83.7 %, respectively. The recovery of internal standard was 100.1%. Precision (% CV) for intra-batch and inter-batch ranged from 1.82 to 3.55 % for all the QC levels. The detailed results are presented in Table 1. The accuracy re-sults for intra-batch and inter-batch were within 94.50

99.35 % at all QC levels (Table 1).

3.2.3. Ruggedness

One complete precision and accuracy batch was proc-essed and analyzed by different analysts using different col-

Fig. (1). Mass spectrum of: (A) TBS precursor ion (protonated precursor [M+H]+ ions at m/z 226.19); (B) IS precursor ion (protonated pre-

cursor [M+H]+ ions at m/z 260.34 as); (C) TBS product ion (major fragmentated product ion at m/z 152.12); and (D) IS product ion (major

fragmented product ions at m/z 183.11) showing fragmentation transitions

UHPLC/ESI-Q-TOF-MS Assay of Terbutaline Current Pharmaceutical Analysis, 2012, Vol. 8, No. 2 5

umn and different sets of solutions. The mean accuracy (n = 6) for drug ranged from 99.0 100.13 and the precision ranged from 1.80 3.06 (Table 2).

3.3. Matrix Effect

Matrix effect is due to co-elution of some endogenous components present in biological samples. The matrix effect (A/B 100) for TB at LQC was 96.01 % (n = 6; % CV 3.28), however at HQC was 97.21 (n = 6; % CV 3.15). The CV (%) < 5 suggested that the method was free from matrix effect. No significant ion suppression or enhancement from plasma

matrix was shown for TB in post-column infusion experi-ments with liquid–liquid extraction (LLE). Various precipita-tion agents were tried (tricholoroacetic acid, trifluoroacetic acid and formic acid). However, when a simple protein pre-cipitation with formic acid was used for sample preparation, a significant ion enhancement was observed at the retention time of 1 min.

3.4. Ex Vivo Stability

First time here we reported the stability evaluation of analytes, actually designed to cover anticipated conditions of

Fig. (2). Typical chromatograms of TB and IS in Wistar rat plasma by selective reaction monitoring scan mode. (A) extracted TB (50 ng

mL-1

); (B) IS (100 ng mL-1

); (C) Extracted blank plasma sample; and (D) extracted TB spiked plasma sample (1 ng mL-1

).

Table 1. Precision and Accuracy Data

Intra-Batch Inter-Batch QC

ID

Theoretical

Content (ng

mL-1

) Mean Concentration Observed

(ng mL-1

)a

Accuracy

(%)

CV

(%)

Mean Concentration Observed

(ng mL-1

)a

Accuracy

(%)

CV

(%)

LQC 2 1.89±0.06 94.50 3.17 1.97±0.07 98.51 3.55

MQC 402 395.08±7.2 98.27 1.82 395.61±8.30 98.41 2.09

HQC 800 787.10±21.41 98.38 2.72 794.84±14.62 99.35 1.83

LLOQ 1 0.95±0.02 95.00 2.10 0.97±0.02 97.00 2.06

aMean of 6 replicates at each concentration (n = 6); CV (%): Coefficient of variance (percentage) = standard deviation divided by concentration found 100; Theoretical contents; LQC: 2 ng mL-1; MQC: 400 ng mL-1; HQC: 800 ng mL-1 and LLOQ: 1 ng mL-1.

Table 2. Ruggedness data

QC ID Theoretical Content (ng mL-1

) Mean Concentration Observed (ng mL-1

)a Accuracy (%) CV (%)

LQC 2 1.98±0.05 99.00 2.52

MQC 402 400.13±7.38 99.53 1.84

HQC 800 801.05±14.43 100.13 1.80

aMean of 6 replicates at each concentration (n = 6); CV (%): Coefficient of variance (percentage) = standard deviation divided by concentration found 100; Theoretical contents; LQC: 2 ng mL-1; MQC: 400 ng mL-1; HQC: 800 ng mL-1 and LLOQ: 1 ng mL-1.

6 Current Pharmaceutical Analysis, 2012, Vol. 8, No. 2 Faiyazuddin et al.

handling of the experimental samples. Table 3, summarizes the results of stability experiments, which showed that TB was stable during all storage conditions (long–term, freeze–thaw, bench–top and post processing stability). The analytes stability in plasma sample was investigated at two QC levels (in LQC and HQC). The recovery of the analytes relative to that at time zero is reviewed. After 1 month of storage (Long-term stability) the recovery of TB was 95.23% (LQC), and 95.78% (HQC). After 1, 2 and 3 cycles of freeze–thaw (freeze–thaw stability), TB was recovered in the range of 97.88–98.94% (LQC) and 95.97–99.76% (HQC). After 24 h (bench–top stability) the recovery of TB was 97.35% (LQC), and 97.09% (HQC). The recovery (post processing stability) of TB was 97.88% (LQC) and 97.45% (HQC).

3.5. Pharmacokinetic Investigation In Vivo

The developed and validated UHPLC/ESI-Q-TOF-MS/MS method described above had been applied success-

fully to the pharmacokinetic investigation of TB in Wistar male rats. Fig. (3) shows the profile of the mean TB plasma concentration versus time. Meanwhile, the main pharma-cokinetic parameters for TB are presented in Table 4. The AUC0 t (mean area under the plasma concentration–time curve) for TB was 735.10±102.33 h.ng mL

-1, whereas T0.5

(mean elimination half-life) was calculated as 8.34±1.42 h. Cmax (mean peak plasma concentration) for TB was 258.00±15.32 ng mL

-1 at Tmax (mean peak time) of 1.00±0.18

h.

4. CONCLUSION

UHPLC with QTOF-MS offers improved quality data in terms of increased detection limits, and chromatographic resolution with greater sensitivity. In the present investiga-tion a validated UHPLC/ESI-Q-TOF-MS/MS method for the determination of TB was optimized. This method was sensi-tive enough to monitor the low-dosage PK studies of TB in plasma. The advantages of our developed method over pre-

Table 3. Ex Vivo Stability Data

Condition LQC HQC

Long term stability; recovery (ng) after storage ( 80 °C)

Initial 1.89 ± 0.01 742.5 ± 10.02

1 month 1.81 ± 0.03 (95.23 %) 711.2 ± 12.51 (95.78%)

Freeze–thaw stability; recovery (ng) after freeze–thaw cycles ( 20 °C to 25 °C)

Cycle 0 1.89 ± 0.01 742.50 ± 10.02

Cycle 1 1.87 ± 0.01 (98.94%) 740.11 ± 12.00 (99.76%)

Cycle 2 1.86 ± 0.01 (98.41%) 731.32 ± 10.17 (98.49%)

Cycle 3 1.85 ± 0.01 (97.88%) 712.61 ± 14.15 (95.97%)

Bench top stability; recovery (ng) at room temperature (25 °C)

0 h 1.89 ± 0.01 742.51 ± 10.02

24 h 1.84 ± 0.01 (97.35%) 720.90 ± 8.25 (97.09%)

Post processing stability; recovery (ng) after storage in autosampler (10 °C)

0 h 1.89 ± 0.01 742.51 ± 10.02

24 h 1.85 ± 0.02 (97.88%) 723.62 ± 11.05 (97.45%)

Values (Mean ± SD) are derived from 6 replicates. Figures in parenthesis represent analyte concentration (%) relative to time zero. Theoretical contents; LQC: 2 ng mL-1; MQC: 400 ng mL-1; HQC: 800 ng mL-1 and LLOQ: 1 ng mL-1.

Fig. (3). Plasma conc. vs. time curve of TB (5 mg/kg, oral) in Wistar rats. Each time point is mean±SD (n = 6).

UHPLC/ESI-Q-TOF-MS Assay of Terbutaline Current Pharmaceutical Analysis, 2012, Vol. 8, No. 2 7

vious techniques are the short analysis time (3 min), high sensitivity (LLOQ: 1.0 ng mL) and simple extraction proce-dure. The assay was successfully employed for pharmacoki-netic studies in Wistar rat plasma with acceptable precision, adequate sensitivity and satisfied accuracy. It would be, fur-thermore, applicable for clinical studies.

ACKNOWLEDGMENTS

Md. Faiyazuddin is thankful to Defense R & D Organiza-tion (DRDO), New Delhi, India for providing financial sup-port for the work presented in this paper. The authors are thankful to Dr. G. N. Qazi, the honb’le Vice Chancellor of Hamdard University for providing the facilities of UHPLC and experimental animals. Authors wish to thank Dr. Sayeed Ahmad, Department of Pharmacognosy & Phytochemistry, Faculty of Pharmacy, Hamdard University for his valuable support. Authors acknowledge the support of Netco Ltd. (Tadbun, Hyderabad, India) and Universal Capsules (Mum-bai, India) for providing gift samples of terbutaline sulphate and capsules (hard gelatin; size#3), respectively. Authors wish to extend sincere thanks to Mr. Abdul Rauf for helping and procuring material in this research.

CONFLICT OF INTEREST

Authors have no conflict of interest in this paper. The re-search work was funded by Defense R & D Organization (DRDO), New Delhi, India.

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Received: Septeember 26, 2011 Revised: November 11, 2011 Accepted: November 29, 2011

Table 4. Pharmacokinetic Parameters

Parameter Mean Value (±SD)

AUC0 t (h.ng mL-1) 735.10±102.33

Cmax (ng mL-1) 258.00±15.32

Tmax (h) 1.00±0.18

T0.5 (h) 8.34±1.42

Mean values are derived from Fig.4 by using non-compartmental pharmacokinetic data analysis software (PK Solutions version 2.0TM; Summit Research Services, Montrose,

USA).