Setting System Suitability Parameters for Performance Optimization


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G.U. Journal of Science 22(3): 149-155 (2009)

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Setting System Suitability Parameters for Performance Optimization of GC-NPD Detection for Pesticide Residue Analysis
Osman TĐRYAKĐ1♠, Dilan BAYSOYU2, Gülizar AYDIN2, Emine SEÇER2
1 Erciyes University, Seyrani Agricultural Faculty, Plant Protection Department, 3809, Kayseri, Turkey 2 Turkish Atomic Energy Authority, Sarayköy Nuclear Research and Training Center, Agriculture Unit,
Saray Mah., Atom Cad., No:27, 06983, Saray, Ankara, Turkey

Received: 14.05.2007 Revised: 22.01.2009 Accepted: 04.03.2009

ABSTRACT
System suitability test (SST) is a test to determine the suitability and effectiveness of chromatographic system prior to use. The performance of any chromatographic system may continuously change during their regular use, which can affect the reliability of the analytical results. The operation parameters of the whole chromatographic system can be checked with properly selected SST mixtures. These mixtures are used to establish characteristic chromatographic parameters, such as the number of effective theoretical plates, resolution, asymmetry, detection limit and selectivity. The system is then only declared suitable if the responses are within given limits. This study was performed to set system suitability criteria for detectability in gas chromatography-nitrogen phosphorus detection (GC-NPD) methods for pesticide residue analysis. Under our laboratory and analysis conditions, asymmetry and tailing of dimethoate and tributylphosphate (TBP) peaks slightly changed after 63 standard and/or sample injections; however these changes were within the required limits.
Key Words: System Suitability Test (SST), GC Performance, GC Optimization.

1. INTRODUCTION
The purpose of the residue analytical laboratories is the fact that the produced data should be reliable. As an internal quality control measure, it is crucial to verify that the chromatographic system fits this purpose. SST which in general is performed at the beginning of and sometimes during the routine analysis, is used to test the whole chromatographic system performance. Thus, SST is an integral part of analytical procedures and is used as part of the internal quality control of the method and its criteria should be established during the method validation [1, 2-5].
Once the method is validated there is no guarantee that the same instrument/conditions will be used to perform the method each time. The changes of chromatographic system performance increase the uncertainty of chromatographic measurements and may result systematic error. Therefore, before the instrumental
♠Corresponding author, e-mail: [email protected]

analysis, performance parameters should be checked to verify their suitability for the analysis [1, 5].
The SST mixture is used for the NPD optimization. The selectivity and sensitivity of NPD depends on H2 flow and the bead temperature, respectively. Tailing of dimethoate and TBP peaks might indicate column contamination and poor NPD bead condition. The dimethoate asymmetry is most sensitive indicator of system contamination. Due to its sensitivity to the operating conditions, it indicates even slight deterioration of the system. Dimethoate can only be reproducibly analyzed with inert and properly installed injector and column. The poor system condition can be rectified by cutting the column and replacing the liner. Therefore, dimethoate can be included in a test mixture as indicators of wrong cut or installation of the column. The number of effective theoretical plates (Neff)

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G.U. J. Sci., 22(3):149-155 (2009)/ Osman TĐRYAKĐ, Dilan BAYSOYU, Gülizar AYDIN, Emine SEÇER

indicates significant changes depending on carrier gas flow and injector port configuration [1, 6].

The resolution of two peak pairs of

quinalphos/methidation

and

chlorpyrifos-

methyl/parathion-methyl are used to illustrate the

separation power of the column with 0.25 and 0.32 µm

film thicknesses, respectively [1].

The present work describes the application of a SST for performance optimization of GC-NPD. The methodology presented in this paper is taken from Soboleva and Ambrus[1], who developed the system suitability test for GC.

2. THEORETICAL

Typically, the SST involves performance parameters, such as Neff, resolution, asymmetry, retention time, detection limit and selectivity. Although most of the performance parameters are calculated automatically by data evaluation software of sophisticated chromatography equipment, their theories are very important. The following definitions of SST parameters and the calculation of them are taken from the various literatures [1, 2, 5, 6-9].

2.1. Response Factor (RF)

RF (mm s/pg), also called sensitivity, is calculated with the following equations (Eq.1 and 2).

RF = Area

CA

(1)

where, CA is the amount of the injected specific atom.

C = nAW C A M r (2)

where, n is the number of carbon or hetero atoms in the molecule, Aw is atomic mass of P, N, S or carbon, C is the amount (pg) of the injected molecule, Mr is molecular mass.

2.2. Detection Limit (LD)

LD (pg/s) is defined as (Eq. 3):

LD = fN

RF

(3)

where, N is the noise level in mm and f is multiplication factor.

2.3. Selectivity (SI)

Eq. (4) is applied for the calculation of selectivity of P and N to carbon:

SI = RF

RFC

(4)

where, RF is the response factor of P and N, and RFC is the response factor of carbon. SI values of both P and N to carbon should be higher than 20000 [7].
2.4. Retention Time (tR)
The tR may vary over time due to a number of causes such as, degradation of column performance, change of column, differences between batches of the mobile phase, variation of ambient temperature. For this reason, it is impossible to give an absolute retention time, rather an acceptable range of retention time is given [2]. The relative retention time (RRT) – its reproducibility is better than that of tR – is defined as the ratio of the retention times of any component (Eq.5) to the retention times of reference compound (relatively stable).

RRT = t R(i) t R (ref ) (5)

where, tR(i) and tR(ref) are the retention times of component i and the reference compound chlorpyrifos,
respectively.

2.5. Resolution (Rs)

Rs is a measure for the ratio of the distance of two adjacent peak maxima and their widths. For complex sample mixtures Rs should be determined for the critical pairs of component to characterize their separation. The Rs of two components is calculated with Eq.6.

R = 2(tR2 − tR1 ) s Wb1 + Wb2 (6)

where, tR2 and tR1 are the retention times of closely eluted compounds, and Wb1 and Wb2 are their peak widths at the base.

In United States Pharmacopoeia (USP) the peak width at the base (Wb), while in Europe and Japan mostly the peak width at half the height (W1/2h) is used [2, 7]. Definitions and calculations were done based on peak
width at the base in this work.

2.6. Theoretical Plate Numbers (Neff)

The efficiency of column is expressed quantitatively by the number of plates. This can be calculated in various ways using different measures for peak broadening. In addition to the plate number determined, very often the plate number/meter values are also given. In the SST report sometimes the height equivalent to a plate [10] values are also indicated. Number of effective theoretical plates is defined in Eq. (7).

N

= 16

t R'

2 

eff

 Wb 

(7)

where, t'R is the adjusted retention time of solute (t'R = tR(i)-t0 ; tR(i) is retention times of component i, t0 is the

G.U. J. Sci., 22(3):149-155 (2009)/ Osman TĐRYAKĐ, Dilan BAYSOYU, Gülizar AYDIN, Emine SEÇER

151

dead time measured by injecting a non-retained compound to the system), Wb is the peak width at the base in seconds. Although the minimum accepted value of Neff for S-ethyl dipropylthiocarbamate (EPTC) and phosalone are given in some literature [7], as 4000 and 20000 per meter of column, respectively, the values of Neff mainly depends on the actual task.

2.7. Asymmetry Factor (As) and Tailing (T)

The performance of a column depends not only on the number of plates but also on the shape of the peaks separated. Skewed, asymmetrical peaks may overlap resulting in decreased resolution. The tailing of a peak can have a major impact on the performance of a quantitative method. Depending on the properties of the column, the sample matrix and the analytes, peak asymmetry may vary over the lifetime of the column. Asymmetry of a peak is given by comparing the fronting and tailing end of the peak. The calculation of As and T are given in Eq. 8 and Eq.9, respectively.

A =b

sa

(8)

T = a+b

2a

(9)

where, a is a front part and b is the back part of the line parallel to base line at the 10 and 5 % of the peak height, for asymmetry and tailing calculations, respectively.

The limit or acceptable range of As is reported by Anonymous [7], Matysova et al [9], Anonymous [11] as
0.7-1.8, <1.5, and 0.8-1.5, respectively. A limit also was given for a tailing, T ≤ 2.5 [7].

The calculated performance parameters for SST, described above, should be within the required limits, otherwise the analysis can not be started.

3. EXPERIMENTAL

3.1. Chemicals

The docosane (hydrocarbon, C22H46) and isooctane were obtained from Merck, with the purity of 98.0 and 99.5%, respectively. All other pesticide active ingredients, purchased from Dr. Ehrenstorfer Laboratories GmbH, Germany, were listed with their purities in Table 1.

3.2. Chromatographic Conditions

Hewlett Packard (HP 6890 Agilent) GC equipped with autosampler (HP 7683) and capillary column connected through a NPD system were used at the following conditions: capillary column (30.0 m length x 320 µm id x 0.25 µm nominal film thickness, HP 19091S-433, HP-5MS 5% Phenyl Methyl Siloxane) ; carrier gas nitrogen 2.0 mL/min, hydrogen 3.0 mL/min; air 60.0 mL/min. Operating Conditions; Column temperature:

70-270 oC; initial time 1 min at 70 oC; rise (I): 20 oC/min to 160 oC – 0 min, rise (II): 4 oC/min to 270 oC – 10 min, total run time: 43 min; detector temperature: 300 oC, injector temperature: 200 oC (splitless), injection volume: 1 µL.
3.3. System Suitability Test Mixtures
The SST mixtures contained 9 compounds which cover the expected retention time range and were suitable to control the performance of the GC coupled with NPD [1]. The composition of GC-SST mixture, which were prepared in isooctane, for monitoring performance of chromatographic system with NPD and the purpose of the use of its components were given in Table 1 [1, 6, 7]. The mixture was injected to GC-NPD in five replications under operating conditions mentioned above, and concentrations indicated in Table 1.
4. RESULTS AND DISCUSSION
System suitability parameters were measured so as to verify the system performance. All important characteristics including the number of effective theoretical plates, resolution, asymmetry, retention time, detection limit and selectivity were measured and calculated by using SST solution injection in five replicates. The results of SST test were also compared with the limits mentioned in literature [7, 9].
Figure 1 shows the comparison of peak tailing and broadening and retention time shift, between initial injection and after 63 injections; including dichlorvos, chlorpyrifos and malathion injections as calibration in sample matrix (0.25 and 2.5 g/mL sample equivalent) and solution, and fortified sample extract as well. Peak tailing and broadening are the first symptomps when the system becomes contaminated [1].
Due to the system contamination, the differences in peak shapes of EPTC, TBP, chlorpyrifos (relatively stabile compounds) and dimethoate, between initial injection and after 63 injections, were illustrated in Figure 2. As can be seen from the figure, peak shapes, tR and RRT values demonstrate that there was no significant differences between initial injection and after 63 injections for all 4 compounds. Though asymmetry and tailing factors for dimethoate and TBP slightly changes after 63 injection, these were within the limit set by some workers [7, 9, 11]. The differences of EPTC peak area was also shown in Figure 2, but the peak areas obtained at different occasions are not comparable, since the sensitivity of the detector also is changing in time [1].
Table 2 presents the average, standard deviation (SD) and relative standard deviation (RSD) of RRT of 5 consequent injections of SST mixture into HP 6890 Agilent GC system. The differences in average RRTs of the components of SST mixture injected at the initial time of analysis and after 63 sample and/or standard injections were also included in the last column of the table. The table shows that the change in GC system performance is best indicated with RRT shift of dimethoate and primicarb.

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G.U. J. Sci., 22(3):149-155 (2009)/ Osman TĐRYAKĐ, Dilan BAYSOYU, Gülizar AYDIN, Emine SEÇER

Table 1. The composition of SST mixtures for GC-NPD and the functions of their components.

Compound
EPTC TBP Dimethoate Pirimicarb Chlorpyriphos ethyl* Quinalphos Methidation Docosane Phosalone *Reference compound

Purity (%)
96.5 99.7 98.5 99.1 98.5 99.5 99.0 98.0 98.5

Molecular formula
C9H19NOS C12H27O4P C5H12NO3PS2 C11H18N4O2 C9H11Cl3NO3PS C12H15N2O3PS C6H11N2O4PS3 C22H46 C12H15CINO4PS2

Concentration, ng/µl
2.01 0.10 0.20 2.01 0.10 0.10 0.10 40.1 0.20

Retention time (tR), min
6.97 10.53 12.03 14.03 17.09 18.98 19.55 22.48 28.64

Purpose of use
Neff, sensitivity Sensitivity, selectivity P to C Asymmetry Sensitivity, selectivity N to C Reference for RRTs Resolution with methidathion Resolution with quinalphos Selectivity Neff, RRT

Pirimicarb

EPTC

(A)

Phosalone

Chlorpyrifos QMueitnhialdpathhoison

TB P Dimethoate

Pirimicarb

EPTC

Phosalone

Chlorpyrifos Quinalpho
Methidsathion

TB P Dimethoat e

(B)
7.5

10

12.5

15

17.5

20

22.5

25

27.5

min

Figure 1. Chromatogram of SST mixture for NPD: (A) initial injection, (B) after 63 sample and/or standard injection.

Table 2. The RRTs of SST compounds in GC system (average, standard deviation-SD and relative standard deviationRSD, %, n=5).

Compounds
EPTC Tributylphosphate Dimethoate Pirimicarb Chlorpyriphos ethyl* Quinalphos Methidation Phosalone

Initial
Average RRT 0.4078 0.6158 0.7034 0.8206 1.0000 1.1099 1.1434 1.6747

SD
0.00002 0.00006 0.00007 0.00002 0.00000 0.00006 0.00012 0.00008

RSD (%) 0.006 0.011 0.009 0.003 0.000 0.006 0.011 0.005

After 63 injection

Average RRT
0.4078 0.6158 0.7035 0.8362 1.0000 1.1099 1.1436 1.6748

SD
0.00003 0.00005 0.00007 0.00004 0.00000 0.00010 0.00007 0.00011

RSD (%) 0.008 0.009 0.010 0.005 0.000 0.009 0.006 0.006

Diffrences
of RRT 0.00004 0.00004 -0.00005 -0.0157 0.00000 -0.00005 -0.0002 -0.00004

G.U. J. Sci., 22(3):149-155 (2009)/ Osman TĐRYAKĐ, Dilan BAYSOYU, Gülizar AYDIN, Emine SEÇER

153

1

Rt

RRt

Asymmetry

Tailing

1

6.975

0.408

1.185

1.133

2

2

6.972

0.408

0.83

0.95

EPTC

2

1

DIMETHOATE

1

TBP

2

Rt

RRt

1

12.030

0.704

2

12.025

0.703

Asymmetry
1.391 1.21

Tailing
1.211 1.14

Rt

RRt

1

10.532

0.616

2

10.525

0.616

Asymmetry
1.428 1.25

Tailing
1.406 1.08

Rt

RRt

Asymmetry

Tailing

1

17.100

1

2

17.094

1

1.142 1.33

1.080 1.21

1

CHLORPYRIFOS

2

Figure 2. Chromatographic peak shapes: (1) SST mixture injection at the begining, (2) SST mixture injection after 63 standard and/or sample injection.

Although chlorpyriphos-methyl and parathion-methyl are well resolved in columns with 0.32 µm film thickness, they co-elute with 0.25 µm film thickness. Therefore, quinalphos /methidathion are used to characterize the resolution power of the 0.25 µm film thickness column (Figure 1 and Table 3).
The calculations and interpretation of performance parameters with NPD SST were summarized in Table 3. The detail of abbreviations and/or their symbol were given in the material and method section or footnote of the table. The selectivity (SI) to P was measured using

the ratio of the RF of phosphorous in TBP to carbon in docosane. Calculated SI of P and N (in primicarb) to carbon were bigger than set value [7]. As an indication of efficiency of column, the number of effective theoretical plates (Neff), was calculated based on peak width at the base with the Eq. (7). Our findings were in accordance with required limits [7], which were 4000 and 20000 per meter of column, for EPTC and phosalone, respectively. The results in Figure 2 and Table 3 indicated that asymmetry and tailing values were also within the limits.

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G.U. J. Sci., 22(3):149-155 (2009)/ Osman TĐRYAKĐ, Dilan BAYSOYU, Gülizar AYDIN, Emine SEÇER

Table 3. Summary of calculated/observed SST performance parameters for GC-NPD system.

Compound nCg/µ, l Mr Spaetocmific nCga/µ,l tR, s Area

RF pLgD/s, SI to C Wsb, Neff As TT Rs

EPTC

2.01

TBP

0.10

Dimethoate 0.20 Pirimicarb 2.01

Chlorpyriph 0.10 os Quinalphos 0.10
Methidation 0.10
Docosane 40.1

Phosalone 0.20

189 266 P
229 238 N
351

418
0.01 632 2
722
0.47 842 2
1026

298

1138

302 N, P, S

1173

310 C

34.1 1349

6

368

1718

320.2 193.8 16.635

0.492

0.048 11037

6.853 1159887 0.087

249.1

1.4

639.6 1.354 69.46 94413 0.097

3

145.9

365.2 111.5 0.49 1.43E-5

37.4

19039

1.2 4.3

Abbreviations: C is the concentration of compound; Mr is the molecular mass; Ca is the concentration of specific atom; tR is the retention time in seconds; RF the response factor; LD limit of detection; SI the selectivity; Wb is the peak width at the base in seconds; Neff is the number of effective theorital plates; As asymmetry factor; T is the tailing of peak, and Rs the resolution.

5. CONCLUSION
SST provided proof that an analysis has been performed consistently and correctly over time, and gave an indication of when a component of the system, such as the column, injector port and detector was fit for the purpose. In pesticide residue analysis, screening for over 150-200 compounds, with largely differing chromatographic properties, has to be performed on a daily basis. As part of quality control measures it is crucial to verify that the GC system fits for the purpose, or if any deterioration occurred during its previous use. This can be most economically done with the injection of SST mixture that can give information for all performances with one injection. The SST mixture, consists of several pesticides to enable analyst, assess whether the GC-NPD systems is fit for its purpose. The critical performance parameters prevailed during the validation of the method should be recorded and reported together with the validation data. If the results of performance parameters are not fit with the required limits, the acceptable operation conditions have to be restored before the analysis. In this study, the most important performance parameters, such as asymmetry and tailing factor for dimethoate and TBP, were found within the set limits.
REFERENCES
[1] Soboleva, E., Ambrus, A., “Application of a system suitability test for quality assurance and performance optimization of a gas chromatographic system for pesticide residue analysis”, Journal of Chromatog, A: 1027, 55-65, (2004).

[2] Szepesy, L., “Fundamentals of Chromatographic Separation, 7.System Suitability Test (SST)”, FAO/IAEA/SIDA/RENPAP Training workshop on Analysis of Pesticide Formulations, Suweon Republic of Korea (1997).
[3] Hund, E., Heyden, Y.V., Massart, D.L., Verbeke, J.S., “Derivation of system suitability test limits from a robustness test on an LC assay with complex antibiotic samples”, Journal of Pharmaceutical and Biomedical Analysis, 30: 1197-1206 (2002).
[4] “Validation of Analytical Procedures, ICH Topic Q2B Methodology. Step 4, Consensus Guideline”, the International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human use (1996).
[5] Coleman, J., Wrzosek, T., Roman, R., Peterson, J., Mcallister, P., “Setting system suitability criteria for detectability in high-performance liquid chromatography methods using signal-to-noise ratio statistical tolerance intervals”, Journal of Chromatog, A (917): 23-27 (2001).
[6] “GLC Laboratory Exercise, Training Workshop on Introduction to QC/QA Measures in Pesticide Analytical Laboratories”, Training and Reference Center for Food and Pesticide Control, Seibersdorf, Vienna, Austria (2002).

G.U. J. Sci., 22(3):149-155 (2009)/ Osman TĐRYAKĐ, Dilan BAYSOYU, Gülizar AYDIN, Emine SEÇER

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[7] “SST Templates in TW2005_Lectures / 2nd week/l/GC Practical”, FAO/IAEA Workshop on Introduction to QC/QA Measures in Pesticide Residue Analytical Laboratories, Seibersdorf, Vienna, Austria (2005).
[8] Obali, M., “Gaz Kromatografisi Sıvı Kromatografisi Đnce Tabaka Kromatografisi”, TMMOB Kimya Mühendisleri Odası, Bilgi Dizini No: 12.
[9] Matysova, L., Solich, P., Marek, P., Havlikova, L., Novakova, S.J., “Separation and determination of terbinafine and its four impurities of similar structure using simple RP-HPLC method”, Talanta, 68: 713-720 (2006).
[10] Li, Y., Chen, M., Chou, G., Wang, ZT., Hu, ZB., “Ruggedness/robustness evalua-tion and system suitability test on United States Pharmacopoeia XXVI assay ginsenosides in Asian and American ginseng by high-performance liquid chromatography”, Journal of Pharmaceutical and Biomedical Analysis, 35: 1083-1091 (2004).
[11] “European Pharmacopoeia”, third ed., Supplement, European Pharmacopoeia, Strasbourg, 30-33 (2001).

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Setting System Suitability Parameters for Performance Optimization