Dynamic Article LinksC<AnalyticalMethods

Cite this: Anal. Methods, 2012, 4, 2081

www.rsc.org/methods PAPER

Publ

ishe

d on

16

May

201

2. D

ownl

oade

d by

Uni

vers

ity o

f W

arsa

w o

n 29

/10/

2014

09:

28:1

2.

View Article Online / Journal Homepage / Table of Contents for this issue

Multi-response optimization of sequential injection chromatographic methodfor determination of lisinopril and hydrochlorothiazide

Abubakr M. Idris,*a Salih A. Naheid,b Rafea E. E. Elgorashe,c Mohamed A. H. Eltayebd

and Ahmed O. Alnajjarc

Received 10th December 2011, Accepted 23rd March 2012

DOI: 10.1039/c2ay05876f

Recently, a more efficient sequential injection chromatograph (SIC) with a high pressure selection valve

has been developed at our laboratory. In the current work, the newly developed SIC systemwas exploited

to optimize and validate a new method for the separation and quantification of lisinopril and

hydrochlorothiazide in pharmaceutical preparations. A multi-response optimization study was

conducted to screen the effect of mobile phase composition on resolution, reagent consumption,

retention time, peak symmetry, peak height and baseline. The factorial design approachwas adopted and

the effect factors were determined. The SIC method has proven to be a competitor to high performance

liquid chromatographic methods with respect to sample frequency, reagent consumption and safety for

the environment, besides instrumentation benefits with respect to inexpensiveness, simplicity and

portability. Short C18 monolithic columns (30 � 4.6 mm) were used to offer a rapid and reagent-saving

procedure. Miniaturized fiber optic spectrometric devices were coupled with the SIC system to provide

more instrumentation portability. Satisfactory separation, peak symmetry and theoretical plates were

achieved. The SIC method was also accurate (the recovery range was 98.8–101.8%), precise (the RSD

range was 0.95–2.29) and sensitive (the limits of detection were below 1.5 mg mL�1).

Introduction

Pharmaceutical preparations of new combinations as well as the

requirements of modern industrial-scale pharmaceutical analysis

encourage researchers to develop new and efficient methods for

multi-quantification including separation procedures. Rapidity,

selectivity, sensitivity and safety for the environment, in addition

to inexpensiveness in terms of reagent consumption, manpower

and instrumentation cost, are all nowadays required when

developing new analytical methods.

FI techniques apply automated, miniaturized and inexpensive

analytical procedures. To date, the family of FI techniques

includes three generations and five versions.1,2 However, the

second generation, which is termed sequential injection analysis

(SIA), is the most effective technique.3 Nevertheless, imple-

menting simultaneous quantification with separation procedures

in FI techniques was a challenge.

Since their introduction, monolithic columns have been

installed in SIA systems. This development has generated a new

version termed sequential injection chromatography (SIC).4 The

aDepartment of Chemistry, College of Science, King Khalid University,P.O. 9004, Abha 61321, Saudi Arabia. E-mail: abubakridris@hotmail.combAtomic Energy Council, Sudan Academy of Science, Khartoum, SudancDepartment of Chemistry, College of Science, King Faisal University,Hofuf, Saudi ArabiadSudan Atomic Energy Commission, Khartoum, Sudan

This journal is ª The Royal Society of Chemistry 2012

bimodal pore structure of monolithic columns, i.e. macro- and

mesopores, allows a higher performance separation at higher

flow rates than that applied in particle columns. The remarkable

advantages of the instrumentation of SIC over that of high

performance liquid chromatography (HPLC) are inexpensive-

ness, portability, ease of use and lower maintenance cost, besides

reduction in reagent consumption and thus better safety for the

environment.

In contrast, because it is a newly proposed technique, SIC has

suffered from some drawbacks. The syringe pump that is

installed in commercial SIC systems has a limited pressure, about

360–435 psi, which limits the flow rates.5 In addition, the syringe

pump has so far a limited volume, namely 4 mL. Moreover,

satisfactory software has not been developed yet for chromato-

graphic calculations. The commercially available software that

has been developed by the manufacturer of SIC systems could be

used for only controlling the system and collecting data.

Furthermore, the selection valve (SV) installed in commercially

available SIC systems has a limited pressure tolerance, which is

250 psi. This drawback causes the problem of solution leakage

especially when applying high flow rates, adopting a mobile

phase including a buffer or installing long monolithic columns.

Despite those limitations, SIC has proven to be powerful in

pharmaceutical analysis.6–10 Moreover, in our previous work,9,10

the challenge of a lower pressure SV has been overcome by

installing a new SV with a higher pressure tolerance (5000 psi).

Anal. Methods, 2012, 4, 2081–2087 | 2081

Publ

ishe

d on

16

May

201

2. D

ownl

oade

d by

Uni

vers

ity o

f W

arsa

w o

n 29

/10/

2014

09:

28:1

2.

View Article Online

The new SV has also the advantage of additional ports. This

development permits analyzing more solutions with a one-shot

programmable analysis.

On the other hand, to gain the maximum efficiency of

analytical methods, critical optimization processes are required.

Towards this end, due to its familiarity, the univariate approach

has been extensively applied for optimizing analytical methods.

Nevertheless, the univariate approach is not always effective

because it optimizes the conditions one by one. This feature

makes the optimization process time-consuming, besides it does

not consider the effect of interactions between conditions. Che-

mometrics, as a multivariate optimization tool, provides the

maximum efficiency of analytical methods in a short period of

time. Hence, chemometrics saves time, minimizes effort and

reduces the consumption of reagents and samples. Despite these

benefits, few SIA and SIC methods were optimized by chemo-

metrics. Recent papers on this topic are available elsewhere.8,10–18

On the other hand, multi-response optimization is desirable in

developing analytical methods including the separation proce-

dure because more than one analytical feature has to be devel-

oped simultaneously.19–23

Lisinopril (LSP) is chemically named (S)-1-[N2-(1-carboxy-3-

phenylpropyl)-L-lysyl]-L-proline dehydrate (Fig. 1a). It is a long-

acting, nonsulfhydryl angiotensin-converting enzyme inhibitor.

Fig. 1 Chemical structures of (a) LSP and (b) HTZ.

Fig. 2 Schematic diagram of a sequential injection chromatograph constru

2082 | Anal. Methods, 2012, 4, 2081–2087

It is used for the treatment of hypertension and congestive heart

failure.24 On the other hand, hydrochlorothiazide (HTZ) is

chemically named 6-chloro-3,4-dihydro-2H-1,2,4-benzothiadia-

zine-7-sulfonamide-1,1-dioxide (Fig. 1b). It is a thiazide diuretic

agent. It is widely used for the treatment of both diuretic and

hypertensive clinical indications.25 The combination of LSP and

HTZ is common in antihypertensive therapy. This combination

reduces blood pressure and hence can minimize the risk of

damage to kidneys, heart or other organs.26

In the literature, many methods have been proposed for the

separation and simultaneous quantification of LSP and HTZ. In

this regard, some HPLC methods have been proposed.27–30 In

those methods, different compositions of mobile phase were used

including isocratic acetonitrile–water (20 : 80, v/v) at pH 3.8.26 A

gradient separation by acetonitrile–25 mmol L�1 phosphate

(7 : 93, v/v) at pH 5.0 and acetonitrile–25 mmol L�1 phosphate

(50 : 50, v/v) at pH 5.0 was proposed as well.28 In other studies,

isocratic separation by acetonitrile–phosphate (50 : 50, v/v) at

pH 6.529 as well as by 1-hexane sulfonic acid sodium salt, trie-

thylamine, orthophosphoric acid, acetonitrile and methanol30

was developed. On the other hand, capillary electrophoresis31

and high performance thin layer chromatography with densi-

tometry,32 besides spectrophotometry,33 were also exploited for

quantification of LSP and HTZ.

The current paper reports a multi-response optimization

process for developing a new SIC method for the separation and

quantification of LSP and HTZ in pharmaceutical formulations.

The factorial design approach was exploited for the optimization

process. The method was validated and a comparative study with

a previous HPLC method27 was conducted.

Experimental

Instrumentation

The following devices were used to construct an SIC system

(Fig. 2):

(i) A S17 PDP� syringe pump (SP) with a reservoir of 4 mL

was manufactured by Sapphire Engineering (Pocasset, MA,

USA).

cted for separation and simultaneous determination of LSP and HTZ.

This journal is ª The Royal Society of Chemistry 2012

Table 1 Typical sequence of the particular steps of the SIC program controlling separation and quantification of LSP and HTZ (a single circle)

Action description Time (s) Valve position Pump action Flow rate (mL s�1) Volume (mL)

Filling syringe with the mobile phase 7 Check valve in the syringe pump Aspirate 150 1000Column conditioning with the mobilephase

33 Central port in the SV with port-2 Dispense 30 1000

Filling syringe with the mobilephase for elution

20 Check valve in the syringe pump Aspirate 150 3000

Standard/sample loading 4 3–10 Aspirate 10 40Separation with UV detection 101 Central port in the SV with port-2 Dispense 30 3040Total 165 — — — 4040

Table 2 Levels of experimental conditions applied for a 26 full-factorialdesign optimization

Experimental conditions Minimum level Maximum level

Acetonitrile ratio (%) 5 15Phosphate concentration (mmol L�1) 20 30pH 4.0 5.0

Publ

ishe

d on

16

May

201

2. D

ownl

oade

d by

Uni

vers

ity o

f W

arsa

w o

n 29

/10/

2014

09:

28:1

2.

View Article Online

(ii) A 10T-0179H Cheminert� high-pressure stainless-steel

selection valve (up to 5000 psi) with 10 ports was manufactured

by Valco Instrument Co. (Houston, TX, USA).

(iii) A USB-4000� fiber optic CCD UV/Vis detector and

a DH-2000� deuterium UV light source were manufactured by

Ocean Optics Inc. (Dunedin, FL, USA).

(iv) An Ultem� 10 mmmicro-volume Z-flow cell was supplied

from FIAlab Instruments Inc. (Bellevue, WA, USA).

(v) 200 micron sub-miniature version A� fiber optic connec-

tors with a core diameter of 600 mm were fabricated by Ceram-

Optec (East Longmeadow, MA, USA).

(vi) Pump tubing of 0.030 0 I.D. Teflon type was supplied from

Upchurch Scientific, Inc. (Oak Harbor, WA, USA). It was used

to connect various devices composing the SIC manifold and to

make a holing coil (200 cm long).

(vii) Chromolith� reversed-phase monolithic separation (4.6

� 25 mm) and guard (4.6 � 5 mm) columns were manufactured

by Merck (Darmstadt, Germany).

PC equipped with FIALab Software� for Windows� version

5.9 was supplied from FIAlab (Medina, WA, USA).

Table 3 23 Full factorial design matrix for screening the effect of buffer conmobile phase volume (MPC, mL), resolution (R), baseline (BL), retention timeseparation column (25� 4.6 mm); sample concentration 50 mg mL�1 for each anm

Exp. no. BC ACN% pH MPV R BL

1 + � + 2000 1.99 0.02 + � � 4200 0.00 0.03 + + + 2000 1.29 0.04 + + � 2500 0.82 0.05 � � + 4500 1.17 0.06 � � � 5500 0.00 0.07 � + + 2000 2.00 0.08 � + � 2500 0.76 0.0

This journal is ª The Royal Society of Chemistry 2012

Chemicals and reagents

All chemicals and reagents used in the current study were of

analytical grade quality. Distilled deionized water was used in all

experiments. Methanol, acetonitrile, sodium hydrogen phos-

phate, LSP and HTZ were supplied by Sigma-Aldrich (Tauf-

kirchen, Germany). Carnauba wax, crospovidone,

hydroxypropyl cellulose, lactose, magnesium stearate and tita-

nium dioxide, as excipients possibly found in tablet formulations,

were a generous gift from Salah Factory for Pharmaceuticals

(Khartoum North, Sudan).

Pharmaceutical samples

Zestroric� tablets (10 mg LSP and 12.5 mg HTZ) were prepared

by Astra Zeneca Pharmaceuticals (LP, Wilmington, USA).

Prinzide� tablets (20 mg LSP and 12.5 mg HTZ) were prepared

by Merck Frosst Canada Ltd. (Kirkland, Quebec, Canada).

Zinopril� tablets (5 mg LSP) were prepared by Jazeera Phar-

maceutical Industries (Riyadh, Saudi Arabia). Lisidene� tablets

(20 mg LSP) were prepared by Sandoz GmbH (Kuandi, Austria).

Zestril� tablets (10 mg LSP) were prepared by Astra Zeneca

Limited (Cheshire, United Kingdom). Esidrex� tablets (25 mg

HTZ) were prepared by Novartis Pharma (Basle, Switzerland).

Preparation of standard solutions

A mixed standard solution of HCZ and LSP (1000 mg mL�1) was

prepared by dissolving appropriate amounts directly in water.

Working standard solutions were prepared by dilution using the

mobile phase.

centration (BC), acetonitrile percentage (ACN%), and pH on consumed(tR, min), peak symmetry (PS) and peak height (PH); fixed conditions: C18

nalyte, flow rate 40 mL s�1, sample volume 40 mL and UV detection at 215

tR (min) PS PH

LSP HTZ LSP HTZ LSP HTZ

99 0.39 1.01 1.5 1.5 0.26 0.6129 0.00 1.01 — — 0.00 0.6821 0.38 0.62 1.36 1.36 0.48 1.3641 0.43 0.59 1.35 1.35 0.45 1.5457 0.41 0.87 1.33 1.33 0.15 0.5465 0.00 0.86 — — 0.00 0.6330 0.36 0.61 1.28 1.28 0.54 1.668 0.44 0.62 1.5 1.5 0.36 1.21

Anal. Methods, 2012, 4, 2081–2087 | 2083

Publ

ishe

d on

16

May

201

2. D

ownl

oade

d by

Uni

vers

ity o

f W

arsa

w o

n 29

/10/

2014

09:

28:1

2.

View Article Online

Preparation of pharmaceutical samples

Twenty tablets of each sample were accurately weighed and

powdered. Water was added to appropriate amounts of powder

to extract LSP and HTZ. The obtained solutions were filtered

through Whatmann� filter paper number one and diluted with

the mobile phase in an appropriate way. All solutions were

filtered through a 0.45 mm membrane before injecting into the

SIC system.

SIC procedure

As shown in Fig. 2, the mobile phase was linked with the check

valve that is positioned at the front of the SP’s head. Waste was

disposed through port 1. The guard column, separation column

and a Z-flow cell were linked with port 2. Standard solutions

were linked with ports 3 to 5. Samples were linked with ports 6 to

10. A holding coil was installed between the central valve of an

SV and a port in the SP head. The relief valve was linked with

a port at the bottom of the SP’s head. A rapid protocol

controlling the proposed SIC procedure was programmed as

illustrated in Table 1.

Fig. 3 Main effect factors of the buffer concentration, pH and ACN

ratio composing the mobile phase on (a) resolution, (b) retention time,

and (c) peak height; fixed conditions: C18 column (4.6 � 30 mm); sample

volume 40 mL, flow rate 40 mL s�1 and UV detection at 215 nm.

Results and discussion

Method optimization

To utilize the benefits of chemometrics, it is recommended to

adopt as few conditions as possible. Therefore, instrumental

conditions presumed to be non-interacting were optimized by the

univariate method. On the other hand, chemical conditions, i.e.

mobile phase components, were optimized by chemometrics.

Short C18 monolithic columns (30� 4.6 mm) were exploited to

provide a rapid procedure. Hence, 20–40 mL s�1 has been found

to be a suitable range of flow rate for separation with respect to

column length. At high flow rate, rapid elution and long peaks

were obtained. On the other hand, with respect to peak height

and peak shape, the practicable range of sample volume has been

found to be 40–60 mL. At large sample volumes, peaks were

significantly heightened while acceptable peak shapes were not

obtained. Therefore, to compromise between those parameters,

a flow rate of 40 mL s�1 and a sample volume of 40 mL were set as

the optimum.

On the other hand, the 23 full-factorial design was adopted to

screen the effect of mobile phase composition on some responses.

Primarily, methanol and acetonitrile were examined. The latter

solvent provided better separation than the former one. More

improvement in chromatograms was recorded when phosphate

buffer was added to acetonitrile. Therefore, the 23 full-factorial

design was adopted to screen the effect of acetonitrile percentage,

phosphate concentration and pH. The base 2 stands for the

minimum and the maximum levels of a condition. The power 3

stands for the number of conditions. Before undertaking any

optimization study, it is important to delineate clearly the

boundaries of the examined conditions.

Preliminary studies revealed that the levels which are given in

Table 2 were suitable for the separation and quantification of

LSP and HTZ. As a result of the adopted factorial design, eight

experiments were conducted. The responses adopted in the

current optimization study were resolution (R), peak symmetry

2084 | Anal. Methods, 2012, 4, 2081–2087

(PS), retention time (tR), peak height (PH), baseline and the

consumed volume of the mobile phase. The matrix of the

adopted design and the obtained results are given in Table 3.

It has been found that the adopted conditions significantly

affected tR with consumption of different volumes of the mobile

phase, which are in the range of 2.0–5.5 mL (Table 3). For the

This journal is ª The Royal Society of Chemistry 2012

Publ

ishe

d on

16

May

201

2. D

ownl

oade

d by

Uni

vers

ity o

f W

arsa

w o

n 29

/10/

2014

09:

28:1

2.

View Article Online

baseline response, various levels were recorded. However, all

levels are acceptable (Table 3). Also, acceptable peaks, in terms of

peak symmetry, were obtained using some conditions (Table 3).

On the other hand, the main effect (Ef) of acetonitrile ratio,

phosphate concentration and pH onR, tR and PHwas calculated

using eqn (1).34 ‘‘y(+1)’’ and ‘‘y(�1)’’ are the response values at

the maximum and the minimum levels of a condition, respec-

tively. ‘‘n’’ is the number of variables at the same level, i.e. ‘‘n’’ in

the current study is 4. The obtained results are depicted in Fig. 3:

Ef ¼P

yðþ1Þn

�P

yð�1Þn

(1)

For the resolution response (Fig. 3a), it has been found that

the pH is the most effective condition, which is three-fold more

effective than acetonitrile ratio. Resolution increased as the pH

and acetonitrile ratio increased. For the tR response, different

types and levels of the effect of the three adopted conditions were

recorded (Fig. 3b). Acetonitrile volume positively affected the tRof LSP while it negatively affected the tR of HTZ. In contrast,

buffer concentration positively affected the tR of HTZ while

a little and negative effect was recorded on the tR of LSP.

Moreover, pH positively affected the tR of both drugs at almost

the same level. On the other side, Fig. 3c shows that the most

effective factor on peak height is the acetonitrile ratio while

a relatively insignificant effect was recorded from buffer

concentration and pH. As shown in Table 3, higher tR was

obtained in experiment numbers one (R ¼ 1.99) and seven (R ¼2.00). However, the peaks of both drugs in experiment number

seven are much taller than those obtained in experiment number

one. On the other hand, the best results of the peak symmetry of

both drugs were recorded in experiment number seven as well.

Therefore, the mobile phase composition of 20 mmol L�1

Table 4 Comparative study of conditions and efficiency of the SIC method

Conditions SIC

Sample matrix TabletsSeparation column C18 (4.6 � 25 mm)Mobile phase composition 20 mmol L�1 PHS–ACNFlow rate (mL min�1) 2.4Sample volume (mL) 40CMPVb (mL) 4Total volume of waste production (mL) 4.04Analysis time (min) 2.8Sample frequency (samples per h) 21.4UV detection (nm) 215Resolution 2.0

HCT

Retention time (min) 0.61Peak symmetry 1.18Theoretical plates 821.15Calibration equation PH ¼ 0.0284C + 0.0686Standard deviation of slope 0.0005Standard deviation of intercept 0.0142Correlation coefficient 0.9997Recovery (%) 98.6–100.3Linear range (mg mL�1) 5–40LOD (mg mL�1) 1.4LOQ (mg mL�1) 4.7

a Phosphate–acetonitrile. b Consumed mobile phase volume.

This journal is ª The Royal Society of Chemistry 2012

phosphate–acetnotrile (85 : 15, v/v) at pH 5.0 was considered the

optimum.

Method validation and comparative study

The proposed SIC method was validated according to the

guidelines of the main regulatory agencies, namely International

Conference on Harmonization (ICH) of Technical Requirements

for Registration of Pharmaceuticals for Human Use35 and the

International Union of Pure and Applied Chemistry (IUPAC).36

The obtained results are given in Table 4. The following sections

describe the process of method validation.

Separation efficiency. As shown in Fig. 4 and Table 4,

acceptable resolution between LSP and HTZ was achieved in

both SIC and HPLC methods. However, LSP and HTZ were

separated in a much shorter time using the SIC method than

using a previous HPLC method.27 Short tR in the SIC method

was due to the use of a short column (30 mm) and its monolithic

structure. Moreover, a short column and high flow rate (40 mL

s�1) provided more symmetrical peaks in the SIC method

(Table 4). On the other hand, an acceptable number of theoret-

ical plates were obtained in both SIC and HPLC27 methods.

Linearity. Several mixed standard solutions of LSP and HTZ

were examined. As shown in Table 4, the linearity and the limits

of the Beer’s law obtained from the SIC method are comparable

with the previous HPLC method.27

Accuracy. The accuracy of the proposed SIC method was

statistically evaluated by Student’s t-test. The validated HPLC

method27 was adopted as reference. The accuracy was examined

with a previous HPLC method

HPLC (23)

TabletsC18 (4.6 � 200 mm)

a (85 : 15, v/v) at pH 5.0 ACN–water (20 : 80, v/v) at pH 3.81.0201010.021062132.3

LSP HCT LSP

0.36 5.05 3.381.16 0.43 0.5408.57 1258.1 375.7PH ¼ 0.0099C + 0.0303 — —0.0000 — —0.0054 — —0.9998 0.9999 0.999596.8–101.8 99.33 99.920–80 1–40 1.5–56.00.68 — —2.28 — —

Anal. Methods, 2012, 4, 2081–2087 | 2085

Fig. 4 Chromatogram for 10 mg mL�1 HTZ and 20 mg mL�1 LSP; conditions: C18 column (4.6 � 30 mm); mobile phase composition: phosphate

(20 mmol L�1)–acetonitrile (85 : 15, v/v) at pH 5; sample volume 40 mL, flow rate 40 mL s�1 and UV detection at 215 nm.

Table 5 Application of the SIC method for the separation and quanti-fication of HTZ and LSP in tablet formulation

Trade name

Content(mg)

Recovery(%)

Intra-dayprecision

Inter-dayprecision

LSP HTZ LSP HTZ LSP HTZ LSP HTZ

Zestoric� 10 12.5 101.8 100.3 0.98 1.11 2.09 2.11Prinzide� 20 12.5 97.7 98.6 1.06 1.20 1.89 2.29Lisidene� 20 — 96.8 — 1.27 — 2.09 —Zestril� 10 — 98.7 — 0.95 — 1.97 —Zinopril� 5 — 97.8 — 1.29 — 2.19 —Esidrex� — 25 — 99.7 — 1.06 — 2.24Pu

blis

hed

on 1

6 M

ay 2

012.

Dow

nloa

ded

by U

nive

rsity

of

War

saw

on

29/1

0/20

14 0

9:28

:12.

View Article Online

through pharmaceutical samples as illustrated in Table 5. The

samples were analyzed seven times using the SICmethod and five

times using the reference HPLC method.27 The calculated t-

values (1.53–1.92) did not exceed the tabulated value (2.23) for

the degree of freedom of 10. This indicates that there is no

significant difference in accuracy between the SIC method and

the adopted reference method27 at the 95% confidence level.

Precision. The intra-day precision was evaluated by analyzing

seven solutions of LSP and HTZ obtained from different doses in

tablets (Table 5). The inter-day precision was evaluated by

analyzing the same solutions five times in five consecutive days.

In general, the RSD values were less than 2.29%, indicating

acceptable precision (Table 5).

Limits of detection and quantification. The limit of detection

(LOD) was examined as the concentration of a solute resulting in

a peak height three times the baseline noise level. The limit of

quantification (LOQ) was examined as the concentration of

a solute resulting in a peak height ten times the baseline noise level.

As shown in Table 4, appropriate LOD and LOQ of both drugs in

2086 | Anal. Methods, 2012, 4, 2081–2087

pharmaceutical formulations were obtained. The detectability of

the proposed SIC method was enhanced in terms of peak heights

by the optimization process as well as the use of large sample

volume, high flow rate and a short monolithic column.

Other analytical features. The total analysis time by SIC was

1.27 min (Tables 1 and 4). Hence, the sample frequency of the

SIC method is higher than that of the previous HPLC method27

(Table 4). Higher rapidity of the proposed procedure was due to

the miniaturization of SIC as well as the use of a short column

(30 nm) and its monolithic structure. Furthermore, the minia-

turization of the SIC technique renders the proposed method

reagent-saving and safe for the environment. The total volume of

the consumed solvent, including column conditioning, was

2.0 mL while that in the previous HPLC method27 was 10 mL.

Conclusions

The current study provides a rapid, inexpensive, reagent-saving

and simple SIC method for the quantification of LSP andHTZ in

pharmaceutical formulations. High rapidity was obtained by

four approaches: (a) the miniaturization of SIC, (b) the use of

short guard and separation columns (4.6 � 30 mm), (c) the use

of a separation column with a monolithic structure instead of

a particle structure and (d) the use of high flow rate (2.4 mL

min�1). On the other hand, the method is inexpensive in terms of

instrumentation cost and reagent consumption. Hence, the

proposed SIC method is suitable to be applied to modern

industrial-scale pharmaceutical analysis.

Acknowledgements

Mr Naheid would like to thank the Atomic Energy Council,

Sudan Academy of Science for allowing him to study for the

Masters Degree in Chemistry. Dr Idris also thanks the

This journal is ª The Royal Society of Chemistry 2012

Publ

ishe

d on

16

May

201

2. D

ownl

oade

d by

Uni

vers

ity o

f W

arsa

w o

n 29

/10/

2014

09:

28:1

2.

View Article Online

Department of Chemistry, College of Science, King Khalid

University, where a part of this work was conducted.

References

1 A. M. Idris, Crit. Rev. Anal. Chem., 2010, 3, 150–158.2 A. M. Idris, Crit. Rev. Anal. Chem., 2010, 4, 218–225.3 J. Ruzicka and G. D.Marshall,Anal. Chim. Acta, 1990, 237, 329–343.4 D. Satinsky, P. Solich, P. Chocholous and R. Karlicek, Anal. Chim.Acta, 2003, 499, 205–214.

5 P. Chocholous, L. Kosarova, D. Satinsky, H. Sklenarova andP. Solich, Talanta, 2011, 85, 1129–1134.

6 P. Chocholous, P. Solich and D. Satinsky, Anal. Chim. Acta, 2007,600, 129–135.

7 A. M. Idris, R. E. E. Elgorashe, F. N. Assubaie and A. O. Alnajjar,Chromatographia, 2011, 73, 431–437.

8 A. M. Idris, S. A. Naheid, M. A. H. Eltayeb, R. E. E. Elgorashe andH. N. Al-Akra, J. Liq. Chromatogr. Relat. Technol., 2011, 34, 2256–2270.

9 A. M. Idris and R. E. E. Elgorashe, Chem. Cent. J., 2011, 5, 28.10 A. M. Idris and R. E. E. Elgorashe,Microchem. J., 2011, 99, 174–179.11 S. M. Sultan, A. M. Idris and K. E. Ibrahim, J. Flow Injection Anal.,

2004, 21, 19–24.12 A.M. Idris, F. N. Assubaie and S.M. Sultan,Microchem. J., 2006, 83,

7–13.13 A. M. Idris, J. Pharmacol. Toxicol. Methods, 2077, 56, 330–335.14 A. M. Idris, F. N. Assubaie and S. M. Sultan, J. Autom. Methods

Manage. Chem., 2007, DOI: 10.1155/2007/32470.15 A. M. Idris, J. Anal. Chem., 2010, 65, 36–42.16 A. M. Idris, Chem. Cent. J., 2011, 5, 9.17 A. M. Idris, A. E. E. Ibrahim, A. M. Abulkibash, T. A. Saleh and

K. E. E. Ibrahim, Drug Test. Anal., 2011, 3, 380–386.18 A. M. Idris, R. E. E. Elgorashe and A. O. Alnajjar, J. Anal. Chem.,

2012, 67, 497–503.

This journal is ª The Royal Society of Chemistry 2012

19 A. M. Idris and A. O. Alnajjar, Acta Chromatogr., 2007, 19, 97–109.20 J. Saurina, TrAC, Trends Anal. Chem., 2010, 29, 1027–1037.21 V. Gomez, M. Miro, M. P. Callao and V. Cerda, Anal. Chem., 2007,

79, 7767–7774.22 A. Alnajjar, H. H. AbuSeada and A. M. Idris, Talanta, 2007, 72, 842–

846.23 A. O. Alnajjar, A. M. Idris and H. AbuSeada, Microchem. J., 2007,

87, 35–40.24 S. G. Lancaster and P. A. Todd, Drugs, 1988, 35, 646–669.25 Martindale: The Complete Drug Reference, ed. S. C. Sweetman, The

Royal Pharmaceutical Society, London, 31st edn, 2002, pp. 844–848, 907–911, 962–963.

26 Basic and Clinical Pharmacology, ed. B. G. Katzung, Lange MedicalBooks/McGraw-Hill, 8th edn, 2001, pp. 155–178, 245–264.

27 N. Erk and M. Kartal, Anal. Lett., 1999, 32, 1131–1141.28 D. Ivanovic, M. Medenica, B. Jancic, N. Knezevic, A. Malenovic and

J. Milic, Acta Chromatogr., 2007, 18, 143–156.29 P. Aparna, S. Rao, K. M. Thomas and K. Mukkanti, Anal. Chem.

Indian J., 2008, 7, 7.30 R. T. Sane, G. R. Valiyare, U. M. Deshmukh, S. R. Singh and

R. Sodhi, Indian Drugs, 1992, 29, 558–560.31 S. Hillaert, K. De Grauwe and W. V. Bossche, J. Chromatogr., A,

2011, 924, 439–449.32 A. El-Gindy, A. Ashour, L. Abdel-Fattah and M. M. Shabana,

J. Pharm. Biomed. Anal., 2001, 25, 923–931.33 N. Erk, Spectrosc. Lett., 1998, 31, 633–645.34 E. D. Morgan, Chemometrics: Experimental Design, ACOL, John

Wiley & Sons, London, UK, 1991.35 ICH Harmonized tripartite guideline validation of analytical

procedures: text and methodology Q2 (R1). Parent Guideline datedOctober 27, 1994 (complementary guideline on methodology datedNovember 6, 1996 incorporated in November 2005), http://www.ich.org/LOB/media/MEDIA417.pdf.

36 International Union of Pure and Applied Chemistry, Pure Appl.Chem., 2002, 74, 835–855.

Anal. Methods, 2012, 4, 2081–2087 | 2087