Jordan Journal of Pharmaceutical Sciences, Volume 3, No.

2, 2010

A Simple and Rapid HPLC-UV Method for the Determination of Umckalin, as an

Herbal Marker, in the Cough Syrup of Pelargonium Extract

M. Hudaib 1 , W. B. Aldahasi2, A. Khdair3, R. Salah4, H. S. AlKhatib1, Y. Bustanji1 and M. K. Mohammad1

1
Faculty of Pharmacy, University of Jordan, Amman, Jordan
2
Biovision Company, Riyadh, Saudi Arabia
3
Faculty of Pharmacy, Applied Sciences University, Amman, Jordan
4
Sana Pharmaceutical Research Co., Amman, Jordan

ABSTRACT
A rapid and simple high performance liquid chromatography (HPLC) method with UV detection for the
quantitation of umckalin, as an herbal marker, in Pelargonium extract cough syrup has been developed and
validated. Chromatographic separation was achieved on a reverse phase Phenomenex®-C18 column (5 µm, 25 cm
× 0.5 mm i.d.) using a mixture of acetonitrile and phosphoric acid (pH 2.5), in 25:75 (v/v) ratio, as a mobile
phase at a flow rate of 1 mL/min under ambient conditions and with UV detection at 310 nm. The method,
applied for umckalin quantitation, showed good linearity over the concentration range of 0.334 –1.667 µg/mL,
with a correlation coefficient (r2) of 0.9996. The limit of detection (LOD) and limit of quantitation (LOQ) of
umckalin were found to be 0.0344 and 0.1031 µg/mL, respectively. In addition, the developed HPLC method
showed acceptable values of repeatability and intermediate precision and indicated high levels of method
accuracy. Simplicity and validity of the method make it highly reliable and especially suitable for routine
quality control analysis.
Keywords: Umckalin, Pelargonium sidoides, HPLC analysis, Cough syrup, Herbal product, Herbal marker.

INTRODUCTION Phytochemically, previous reports have led to the

characterization of several metabolites including phenolic
Pelargonium sidoides DC. (family: Geraniaceae) is a and cinnamic acids, tannins, flavonoids, and coumarins in
medicinal plant, with woody roots, that has been used for the different parts of P. sidoides(1,8). The plant extracts,
the treatment of upper and lower respiratory tract diseases obtained particularly from roots, were found to contain
for hundreds of years(1,2). Moreover, antibacterial and substituted and unsubstituted oligomeric prodelphinidins,
immunomodulatory activities have been proven(3,4). monomeric and oligomeric carbohydrates, minerals,
Several commercially available extracts of P. sidoides peptides, purine derivatives, and highly substituted
have clinically and pharmacologically proven efficacy for benzopyranones, which include mainly umckalin (7-
the treatment of cough associated with acute bronchitis or Hydroxy-5,6-dimethoxycoumarin) as the most common
common cold(5,6). Such extracts are commonly called bioactive component of this plant species. Nearly 230
“Umckaloabo”. For example, EPs® 7630 (Umckaloabo®, components have been also detected in the plant essential
Spitzner Arzneimittel) has been developed from extract oil, with sesquiterpenes the most abundant(9).
of P. sidoides and introduced into the European market(7). Despite a wide pharmaceutical use of P. sidoides,
erroneous identification of the raw plant material and
Received on 22/11/2009 and Accepted for Publication on confusion with other species especially P. reniforme is
14/3/2010. problematic to the manufacturing and use of the
E-mail: [email protected]
© 2010 DAR Publishers/University of Jordan. All Rights Reserved.

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Jordan Journal of Pharmaceutical Sciences, Volume 3, No. 2, 2010

plant(10,11). In addition, quantitative analysis in the quality phase Phenomenex®-C18 (5 µm, 25 cm × 0.5 mm i.d.,
control of finished pharmaceutical products is a burden to Thermo Electron Company, Bellefonte, North America).
the manufacturing of such products. Thus, precise Data from each chromatographic run were processed
analytical methods to identify P. sidoides in raw materials using ChromQuest® v 4.1 LC data system (Thermo
and more importantly in the pharmaceutical products are Electron Corporation, USA).
needed to enable safe and effective use of the plant.
Previous studies have reported identification and analysis Methods
of P. sidoides of wild harvested and cultivated plants Preparation of Standard Solutions and Calibration
using HPLC(12). However, up to our knowledge there is Curve
no previous method available for quantitative analysis of A standard stock solution was prepared by
P. sidoides in pharmaceutical products. In the present transferring accurately weighed 2.50 mg umckalin
work we report the development of a simple, sensitive, reference standard into a 50 mL volumetric flask. Then
precise, and specific HPLC method for the analysis of a 40 mL of the diluent (the mobile phase) was added
P. sidoides-based pharmaceutical product (syrup). followed by sonication for 15 minutes or until dissolved.
The method reported here is an isocratic reversed The volume was completed with the same diluent and
phase HPLC assay with UV detection that can be applied then filtered through 0.2 µm membrane filter.
to the determination of umckalin in commercially To study the linearity range of umckalin, serial
available pharmaceutical products of P. sidoides. dilutions of the above standard stock solution were made
Umckalin, the bioactive constituent typical to P. sidoides, to prepare the calibration solutions, which were analyzed
is used as a reliable identifying marker(3,8). by HPLC as described below. A graph was plotted as
concentration of drug versus peak area (response) and
HO O O found linear in the range of 0.334-1.667 µg/mL of
umckalin.
Preparation of Samples
O Four milliliters of the liquid samples (Pelargonium
O syrup or diluted extract) were mixed with 15 mL of the
mobile phase and then sonicated for 15 min. Volume was
Umckalin adjusted to 20 mL with the mobile phase and processed
as described under the standard solution preparation. For
purpose of product assay, three quality control sample
EXPERIMENTAL solutions were prepared according to this procedure with
Materials and Instruments a supposed final concentration of 1 µg/mL umckalin (test
Acetonitrile and phosphoric acid were purchased from concentration). The prepared solutions were analyzed,
Fischer Scientific (Chicago, IL). Pelargonium dried extract twice each, by the developed HPLC method. Further
(standardized to contain 330 ppm umckalin), authentic samples were also prepared for precision assessment as
umckalin reference standard, as well as the Pelargonium will be described below.
syrup (potency: 5 µg/mL umckalin) were a gift from HPLC Method
SANA Pharmaceutical Research Co. (Amman, Jordan). Twenty µL aliquots of standard and sample solutions
A Finnigan™ HPLC system with Surveyor® LC were injected in triplicate into the column and umckalin
Pump Plus and Surveyor® Autosampler Plus connected to was detected at 310 nm wavelength. A mobile phase
Surveyor® PDA Plus detector was used (Thermo Electron consisting of filtered and degassed mixture of acetonitrile
Corporation, San Jose, CA). The column was a reverse and phosphoric acid (pH 2.5) in 25: 75 (v/v) ratios was

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A Simple and Rapid… M. Hudaib et al

used at a flow rate of 1 mL/min under ambient conditions RESULTS AND DISCUSSION
(25 °C). Under these conditions the peak corresponding In the present study a reversed phase HPLC-UV
to umckalin was identified at 11.13 min average retention method for the separation and quantitation of umckalin in
time. a P. dioides extract containing pharmaceutical syrup was
Method Validation developed. The chromatographic conditions were
The HPLC method was validated in terms of linearity, adjusted and optimized to provide reliable and efficient
precision, specificity, limit of detection (LOD), limit of chromatographic and assay performances. In particular,
quantitation (LOQ), and accuracy according to the USP the selection of mobile phase was based on peak
pharmacopoeia and International Conference on parameters, ease of preparation, cost, and total analysis
Harmonization (ICH) guidelines(13). Precision of the time. In the best scenario, separation of umckalin from
assay method was determined using six-independent test other matrix components was successfully achieved in
solutions at concentration of 1 µg/mL. Linearity of the about 12 min total analysis time as shown in Fig. 1.
assay was determined over five different umckalin
concentrations (0.334, 0.667, 1.000, 1.334, and 1.667 Method Validation
µg/mL), each analyzed in duplicate. The concentrations Specificity
of calibration solutions, used in linearity assessment, According to ICH guidelines and some other
were chosen to cover the specified range of the method compendial sources, for chromatographic procedures,
(50%-150% of the test concentration). The response, specificity is usually demonstrated by representative
represented by the average peak area, was plotted against chromatograms where individual components should be
concentration and least square regression analysis was appropriately labeled.
applied. LOD and LOQ of the method were calculated In order to determine the specificity of the present
using the slope (S) of the calibration curve, obtained from HPLC method, the chromatogram obtained from placebo
linearity assessment, and the standard deviation of the analysis (Fig. 1A) was compared with the one obtained
response (SD) calculated as the standard deviation of the after Pelargonium extract syrup analysis (Fig. 1B). As
y-intercepts of regression lines plotted at the shown, a complete separation of the umckalin peak was
chromatographic response values. Specificity of the assay demonstrated without any interference by any other syrup
method was evaluated by injecting Pelargonium syrup component at the retention time of the drug in the
placebo, consisting of all matrix components with the chromatogram of placebo solution. Moreover, in a peak
exception of the dried extract, in duplicate, under the purity analysis, performed using a photo diode array
analytical conditions of the developed method. Precision detector, no peaks appeared at the retention time of
of the method was evaluated by repeatability (intraday) umckalin with a calculated peak purity of 0.9965.
and intermediate precision (interday) tests and evaluated Linearity
by the calculated coefficient of variation (CV), or relative Linearity of an analytical method is defined as its
standard deviation (RSD), where values of less than 2% ability to give test results or a response that is
were considered acceptable. The method accuracy was proportional to the concentration of analyte in specified
evaluated as the percent recovery of the added standard range either directly or by mathematical
(13)
from the accuracy samples, which were prepared by the transformation . The ICH guidelines indicate that a
addition (spiking) of known amounts of umckalin specified range is usually determined according to the
reference standard to the placebo. Accuracy was purpose of the analytical method. For example, the assay
evaluated at five different concentrations (0.333, 0.666, of an active ingredient is usually tested in the range of
1.000, 1.333 and 1.666 µg/mL) of umckalin, analyzed in 80%-120% of test concentration. In our experiment, the
duplicate. selected range was 50%-150% of the test concentration (1

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Jordan Journal of Pharmaceutical Sciences, Volume 3, No. 2, 2010

µg/mL), which corresponded to a range of (0.5-1.5)

µg/mL of umckalin concentration.
A As recommended by the ICH guidelines, the linearity
of an analytical method should be determined at least by
five different concentrations covering the specified range.
As shown in Figure 2, linearity was obtained over the
range of 33.4-166.7% of test umckalin at five different
concentrations (0.334, 0.667, 1.000, 1.334, and 1.667
µg/mL). The Average regression equation over the range
of 0.334 –1.667 µg/mL was y = 882115x -13175 with a
slope of 882115, an intercept of 13175 and a correlation
coefficient (r2) value of 0.9996.
LOD and LOQ
Several approaches are proposed by the ICH
guidelines to determine LOD and LOQ of instrumental
analytical procedures. In the present study these values
B were determined as follows: LOD = 3.3×SD/S, LOQ =
10×SD/S (or LOQ = 3×LOD).
Where, SD = the standard deviation of the response,
and S = the slope of the calibration curve. The slope (S)
was estimated from the linear regression equation
described in the previous section. Particularly, the value
of SD was estimated as the standard deviation of the y-
intercepts of 5 regression lines, each of which is plotted
parallel to the calibration line and passing through the
average chromatographic response of a given calibration
standard. The SD value was further confirmed by another
method based on the residual standard deviation of the
calibration line (data are not given). The values
C corresponding to LOD (0.0344 µg/mL), and LOQ
(0.1031 µg/mL) and the method used for their calculation
are all summarized in Table 1. Noteworthy, the obtained
LOQ value was lower than the lowest value of the
linearity range. Subsequently, the LOQ was validated by
the independent analysis, in duplicate; of 6 samples
containing umckalin in concentration very close to the
quantitation limit (data are not given).
As shown (Table 1), the results indicated that the
current method is suitable to detect and measure very
Figure 1: HPLC chromatogram of (A) Pelargonium extract small product concentrations of umckalin.
syrup placebo, (B) Pelargonium extract syrup, and (C) Precision
Umckalin standard. Precision can be defined as the measurement of

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 A Simple and Rapid… M. Hudaib et al

degree of agreement for a set of test results after recovery percentage for a herbal preparation is considered
repeatable sampling from a homogenous sample. It is to be generally acceptable if it lies within 95%-106% of
usually measured using relative standard deviation (RSD) the true value.
or coefficient of variation (CV) with accepted values to In the present study, accuracy was determined at five
be less than 2%. The precision of the current assay different concentration levels (0.333, 0.666, 1.000, 1.333
method was determined by repeatability (intraday and 1.666 µg/mL) of umckalin, in duplicate, to allow the
precision) and intermediate (interday) precision. calculation of percent recovery values. As shown in Table
Repeatability was evaluated by assaying 6 sample 3, the accuracy results, expressed as mean percent recovery
solutions, prepared at 100% level of the test content (1.0 (± RSD), proved that the developed method is accurate.
µg/mL), during the same day. The intermediate precision
was assessed by comparing the results of repeated assays Product Assay
performed in another day (24 hours apart). The assay results of quality control sample solutions
The data of repeatability and intermediate precision analyzed by the developed method showed an average
investigations, presented in Table 2, demonstrated that content of 99.3% (RSD = 0.8%) of labeled umckalin
the current method can be used for umckalin analysis amount in the tested Pelargonium syrup.
with acceptable level of precision.
CONCLUSIONS
In this study, a reversed-phase HPLC method for the
1600000
analysis of pharmaceutical products (syrups) based on
1400000 y = 882115x - 13175
r 2 = 0.9996
extracts from P. sidoides has been developed and
1200000
validated for the quantitative determination of the
Average peak area

1000000
reference marker, umckalin. Validation was performed
800000
according to the validation protocol of ICH guidelines,
600000
which showed that the developed HPLC assay is simple,
400000
specific, linear, precise, and accurate. The reported
200000
method was very specific as the peak corresponding to
0
0 0.25 0.5 0.75 1 1.25 1.5 1.75 umckalin, the marker, was well separated from the peaks
Concentration (µg/mL) of other product components (impurities and excipients)
Figure 2: Linearity of umckalin response (peak area) over the with a total runtime of 12 min. The method was found
concentration range of 0.334-1.667 µg/mL (the y-error bars linear over the concentration range of 0.334-1.667
represent the standard deviation of the response at each µg/mL. In addition, the current method showed also high
concentration). levels of precision, repeatability, and intermediate
precision and indicated high levels of method accuracy.
Simplicity and validity of the method make it highly
Accuracy reliable and especially suitable for routine quality control
Accuracy could be defined as the closeness of test and analysis.
results obtained by the analytical method to the true
value. According to ICH guidelines, the accuracy of an ACKNOWLEDGEMENT
analytical method should be assessed using a minimum of The authors wish to thank the Deanship of Academic
9 determinations over a minimum of 3 concentration Research (DAR) at the University of Jordan for their
levels covering the specified range of the method(13). The generous funding of this project.

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 Jordan Journal of Pharmaceutical Sciences, Volume 3, No. 2, 2010

Table 1: Calculation and results of limit of detection (LOD) and limit of quantitation (LOQ).
Concentration Limit of detection Limit of quantitation
y-intercept* Standard deviation of the response (SD)
(µg/mL) (LOD)a (LOQ)a
0.334 -9239.41
0.667 -8010.71
1.000 -29227.00 9183.794 0.0344 µg/mL 0.1031 µg/mL
1.334 -12285.40
1.667 -7112.71
*
At each concentration, the y-intercept was calculated from linear regression equation (y = 882115x – 13175) as follows: y-intercept = y – 882115x, where
y: is the average peak area (response) obtained at each concentration.
a
For calculation of LOD and LOQ, see Experimental.

Table 2: Repeatability and intermediate precision tests: Chromatographic peak areas and retention times of
umckalin in Pelargonium extract syrup samples (n = 6) made at a concentration of 1µg/mL.
Sample number 1 2 3 4 5 6 RSD*
Measured peak area (Day 1) 866416 864915 871242 870642 853580 857345 0.828
Retention time 11.14 11.14 11.13 11.13 11.12 11.12 0.08
Measured peak area (Day 2) 873345 870332 868762 874305 865947 868562 0.362
Difference (%) 0.8 0.63 0.28 0.42 1.45 1.31 NA
*
RSD: relative standard deviation; Values of less than 2% are usually acceptable.

Table 3: Accuracy study: Mean percent recoveries of umckalin from placebo samples spiked with known amounts of
umckalin reference standard and analyzed by the developed HPLC method.
Percent of the labeled Added amount of Found amount of umckalin
% recovery*
umckalin content (1µg/mL) umckalin (µg) (µg)
0.333 0.348
Level 1 (33.3%) 105.3
0.333 0.353
0.666 0.693
Level 2 (66.6%) 103.9
0.666 0.691
1.000 1.007
Level 3 (100.0%) 100.6
1.000 1.005
1.333 1.379
Level 4 (133.3%) 103.5
1.333 1.381
1.666 1.716
Level 5 (166.6%) 102.4
1.666 1.696

Mean % recovery ± RSD 103.1 ± 1.7

*
The average value of two determinations; RSD: Relative standard deviation

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Jordan Journal of Pharmaceutical Sciences, Volume 3, No. 2, 2010

REFERENCES controlled, multicentre trial. Planta Med. 2008;

(1) Kolodziej, H. In: Geranium and pelargonium: the 74(6):686-92.
genera Geranium and Pelargonium. Lis-Balchin M. (8) Schoetz, K., Erdelmeier, C., Germer, S., and Hauer H.
(Ed); Taylor & Francis: New York, 2002; Chapter 24, A detailed view on the constituents of EPs 7630.
262-290. Planta Med. 2008; 74(6):667-74.
(2) Timmer, A., Gunther, J., Rucker, G., Motschall, E., (9) Kayser, O., Latte, K., Kolodziej, H., and Hammerschmidt
Antes, G., and Kern W.V. Pelargonium sidoides F. Composition of the essential oils of Pelargonium
extract for acute respiratory tract infections. Cochrane sidoides DC and Pelargonium reniforme Curt. Flavour
Database Syst. Rev. 2008; 16(3):CD006323. Fragrance J. 1998; 13(3):209-12.
(3) Kayser, O. and Kolodziej, H. Antibacterial activity of (10) Brendler, T. and van Wyk, B.E. A historical, scientific
extracts and constituents of Pelargonium sidoides and and commercial perspective on the medicinal use of
Pelargonium reniforme. Planta Med. 1997; 63(6):508-10. Pelargonium sidoides (Geraniaceae). J.
(4) Kayser, O., Kolodziej, H., and Kiderlen, AF. Ethnopharmacol. 2008; 119(3):420-33.
Immunomodulatory principles of Pelargonium (11) Bladt, S, and Wagner, H. From the Zulu medicine to
sidoides. Phytother. Res. 2001; 15(12):122-6. the European phytomedicine Umckaloabo.
(5) Matthys, H. and Heger, M. EPs 7630-solution--an Phytomedicine 2007; 14(6):2-4.
effective therapeutic option in acute and exacerbating (12) White, A.G., Davies-Coleman, M.T. and Ripley, B.S.
bronchitis. Phytomedicine 2007; 14(6):65-8. Measuring and optimising umckalin concentration in
(6) Lizogub, V.G., Riley, D.S., and Heger, M. Efficacy of a wild-harvested and cultivated Pelargonium sidoides
Pelargonium sidoides preparation in patients with the (Geraniaceae). S. Afr. J. Bot. 2008; 74(2):260-267.
common cold: a randomized, double blind, placebo- (13) ICH Harmonised Tripartite Guideline. Validation of
controlled clinical trial. Explore (NY) 2007; 3(6):573-84. Analytical Procedures: Text and Methodology Q2(R1).
(7) Matthys, H. and Funk, P. EPs 7630 improves acute International Conference on Harmonization of Technical
bronchitic symptoms and shortens time to remission. Requirements for Registration of Pharmaceuticals for
Results of a randomised, double-blind, placebo- Human Use: Geneva, Switzerland. 2005.

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‫‪Jordan Journal of Pharmaceutical Sciences, Volume 3, No. 2, 2010‬‬

‫ﺘﻁﻭﻴﺭ ﻁﺭﻴﻘﺔ ﺒﺴﻴﻁﺔ ﻭﺴﺭﻴﻌﺔ ﺒﺎﺴﺘﺨﺩﺍﻡ ﺍﻟﻜﺭﻭﻤﺎﺘﻭﻏﺭﺍﻓﻴﺎ ﺍﻟﺴﺎﺌﻠﺔ ﻋﺎﻟﻴﺔ ﺍﻷﺩﺍﺀ ﻭﺍﻟﻤﻜﺸﺎﻑ ﻓﻭﻕ‬
‫ﺍﻟﺒﻨﻔﺴﺠﻲ ﻟﺩﺭﺍﺴﺔ ﻭﺘﺤﻠﻴل ﻤﺭﻜﺏ ﺍﻷﻤﻜﺎﻟﻴﻥ‪ ،‬ﻜﺩﺍﻟﺔ ﻨﺒﺎﺘﻴﺔ‪ ،‬ﻓﻲ ﻤﻨﺘﺞ ﺸﺭﺍﺏ ﺍﻟﺴﻌﺎل ﺍﻟﻤﺤﺘﻭﻱ ﻋﻠﻰ‬
‫ﺨﻼﺼﺔ ﻨﺒﺎﺕ ﺍﻟﺠﺎﺭﺩﻴﻨﺎ‬

‫‪1‬‬
‫ﻤﺤﻤﺩ ﻫﺩﻴﺏ‪ ، 1‬ﻭﻫﻑ ﺍﻟﺩﻫﺎﺴﻲ‪ ، 2‬ﺃﻴﻤﻥ ﺨﻀﻴﺭ‪ ، 3‬ﺭﺍﺌﺩ ﺼﻼﺡ‪ ، 4‬ﺤﺎﺘﻡ ﺍﻟﺨﻁﻴﺏ‪ ، 1‬ﻴﺎﺴﺭ ﺍﻟﺒﺴﺘﻨﺠﻲ‬
‫‪1‬‬
‫ﻭﻤﺤﻤﺩ ﻤﺤﻤﺩ‬

‫‪ 1‬ﻜﻠﻴﺔ ﺍﻟﺼﻴﺩﻟﺔ‪ ،‬ﺍﻟﺠﺎﻤﻌﺔ ﺍﻷﺭﺩﻨﻴﺔ‪ ،‬ﻋﻤﺎﻥ‪ ،‬ﺍﻷﺭﺩﻥ‪.‬‬

‫‪ 2‬ﺸﺭﻜﺔ ﺒﻴﻭﭭﺠﻥ‪ ،‬ﺍﻟﺭﻴﺎﺽ‪ ،‬ﺍﻟﻤﻤﻠﻜﺔ ﺍﻟﻌﺭﺒﻴﺔ ﺍﻟﺴﻌﻭﺩﻴﺔ‪.‬‬
‫‪ 3‬ﻜﻠﻴﺔ ﺍﻟﺼﻴﺩﻟﺔ‪ ،‬ﺠﺎﻤﻌﺔ ﺍﻟﻌﻠﻭﻡ ﺍﻟﺘﻁﺒﻴﻘﻴﺔ‪ ،‬ﻋﻤﺎﻥ‪ ،‬ﺍﻷﺭﺩﻥ‪.‬‬
‫‪ 4‬ﺸﺭﻜﺔ ﺴﻨﺎ ﻟﻸﺒﺤﺎﺙ ﺍﻟﺼﻴﺩﻻﻨﻴﺔ‪ ،‬ﻋﻤﺎﻥ‪ ،‬ﺍﻷﺭﺩﻥ‪.‬‬

‫ﻤﻠﺨـﺹ‬

‫ﺘﻡ ﻓﻲ ﻫﺫﻩ ﺍﻟﺩﺭﺍﺴﺔ ﺘﻁﻭﻴﺭ ﻭﺇﺜﺒﺎﺕ ﺃﺩﺍﺀ ﻁﺭﻴﻘﺔ ﺘﺤﻠﻴل ﺒﺴﻴﻁﺔ ﻭﺴﺭﻴﻌﺔ ﺒﺎﺴﺘﺨﺩﺍﻡ ﺍﻟﻜﺭﻭﻤﺎﺘﻭﻏﺭﺍﻓﻴﺎ ﺍﻟﺴﺎﺌﻠﺔ ﻋﺎﻟﻴﺔ ﺍﻷﺩﺍﺀ ﻭﺍﻟﻤﻘﻴﺎﺱ‬
‫ﻓﻭﻕ ﺍﻟﺒﻨﻔﺴﺠﻲ ﻟﻠﺘﺤﻠﻴل ﺍﻟﻜﻤﻲ ﻟﻤﺭﻜﺏ ﺍﻷﻤﻜﺎﻟﻴﻥ )‪ ،(Umckalin‬ﻜﺩﺍﻟﺔ )ﻭﺍﺼﻤﺔ( ﻨﺒﺎﺘﻴﺔ‪ ،‬ﻓﻲ ﻤﻨﺘﺞ ﺸﺭﺍﺏ ﺍﻟﺴﻌﺎل ﺍﻟﻤﺤﺘﻭﻱ‬
‫ﻋﻠﻰ ﺨﻼﺼﺔ ﻨﺒﺎﺕ ﺍﻟﺠﺎﺭﺩﻴﻨﺎ )‪ .(Pelargonium‬ﺘﻡ ﺇﺠﺭﺍﺀ ﻋﻤﻠﻴﺔ ﺍﻟﻔﺼل ﺍﻟﻜﺭﻭﻤﺎﺘﻭﻏﺭﺍﻓﻲ ﺒﺎﺴﺘﺨﺩﺍﻡ ﻋﻤﻭﺩ ﻓﺼل ﻤﻌﻜﻭﺱ‬
‫ﺍﻟﻁﺒﻘﺔ ﺍﻟﺜﺎﺒﺘﺔ )‪ (C18‬ﻤﻥ ﺍﻟﻨﻭﻉ ®‪ 5) Phenomenex‬ﻤﻴﻜﺭﻭﻤﻴﺘﺭ‪ 25 ،‬ﺴﻡ ‪ 0.5 x‬ﻤﻠﻡ – ﻗﻁﺭ ﺩﺍﺨﻠﻲ( ﻭﺍﺴﺘﺨﺩﺍﻡ ﺨﻠﻴﻁ ﻤﻥ‬
‫ﺍﻷﺴﻴﺘﻭﻨﺘﺭﻴل ﻭﺤﺎﻤﺽ ﺍﻟﻔﺴﻔﻭﺭﻴﻙ )ﺩﺭﺠﺔ ﺍﻟﺤﻤﻭﻀﺔ ‪ (2.5‬ﻜﻁﺒﻘﺔ ﻤﺘﺤﺭﻜﺔ ﺒﻤﻌﺩل ﺠﺭﻴﺎﻥ ‪ 1‬ﻤل‪/‬ﺩﻗﻴﻘﺔ‪ ،‬ﺘﺤﺕ ﻅﺭﻭﻑ ﻋﺎﺩﻴﺔ‬
‫)ﺤﺭﺍﺭﺓ ﺍﻟﻐﺭﻓﺔ( ﻭﺍﻟﻘﻴﺎﺱ ﺒﻭﺍﺴﻁﺔ ﺍﻷﺸﻌﺔ ﻓﻭﻕ ﺍﻟﺒﻨﻔﺴﺠﻴﺔ ﻋﻠﻰ ﻁﻭل ﻤﻭﺠﺔ ‪ 310‬ﻨﺎﻨﻭﻤﻴﺘﺭ‪ .‬ﺃﻋﻁﺕ ﻫﺫﻩ ﺍﻟﻁﺭﻴﻘﺔ‪ ،‬ﺍﻟﻤﺴﺘﺨﺩﻤﺔ‬
‫ﻓﻲ ﺘﺤﺩﻴﺩ ﻜﻤﻴﺔ ﺍﻷﻤﻜﺎﻟﻴﻥ‪ ،‬ﻤﻨﺤﻨﻰ ﻤﻌﻴﺎﺭﻴﹰﺎ ﺨﻁﻴﹰﺎ ﻟﻺﺴﺘﺠﺎﺒﺔ ﻋﻠﻰ ﻤﺩﻯ ﺍﻟﺘﺭﺍﻜﻴﺯ ﻤﻥ ‪ 0.334‬ﺇﻟﻰ ‪ 1.667‬ﻤﻴﻜﺭﻭﻏﺭﺍﻡ‪/‬ﻤل‬
‫ﻭﺒﻤﻌﺎﻤل ﺍﺭﺘﺒﺎﻁ )‪ (r2‬ﻤﻘﺩﺍﺭﻩ ‪ .0.9996‬ﺃﻅﻬﺭﺕ ﺍﻟﺩﺭﺍﺴﺔ ﻜﺫﻟﻙ ﺃﻥ ﺍﻟﺤﺩﻭﺩ ﺍﻟﺩﻨﻴﺎ ﻟﺘﺭﺍﻜﻴﺯ ﺍﻷﻤﻜﺎﻟﻴﻥ ﺍﻟﺘﻲ ﻴﻤﻜﻥ ﻜﺸﻔﻬﺎ )‪(LOD‬‬
‫ﺃﻭ ﻗﻴﺎﺴﻬﺎ ﻜﻤﻴﹰﺎ )‪ (LOQ‬ﻫﻲ ﺒﻤﻘﺩﺍﺭ ‪ 0.0344‬ﻭ‪ 0.1031‬ﻤﻴﻜﺭﻭﻏﺭﺍﻡ‪/‬ﻤل‪ ،‬ﻋﻠﻰ ﺍﻟﺘﻭﺍﻟﻲ‪ .‬ﺇﻀﺎﻓﺔ ﺇﻟﻰ ﺫﻟﻙ ﺃﻅﻬﺭﺕ ﺍﻟﺩﺭﺍﺴﺔ‬
‫ﻗﻴﻤﹰﺎ ﺠﻴﺩﺓ ﻭﻤﻘﺒﻭﻟﺔ ﻟﻌﻭﺍﻤل ﺍﻟﺩﻗﺔ‪ ،‬ﺍﻟﻤﺘﻌﻠﻘﺔ ﺒﺈﻋﺎﺩﺓ )ﺘﻜﺭﺍﺭ( ﺍﻟﺘﺤﻠﻴل‪ ،‬ﻗﺼﻴﺭﺓ ﻭﻤﺘﻭﺴﻁﺔ ﺍﻟﻤﺩﻯ‪ ،‬ﻭﻜﺫﻟﻙ ﻤﺴﺘﻭﻴﺎﺕ ﻋﺎﻟﻴﺔ ﻟﺼﺤﺔ‬
‫ﺍﻟﻁﺭﻴﻘﺔ‪ .‬ﺒﺴﺎﻁﺔ ﻭﺜﺒﺎﺘﻴﺔ ﺃﺩﺍﺀ ﻫﺫﻩ ﺍﻟﻁﺭﻴﻘﺔ ﺍﻟﻤﻁﻭﺭﺓ ﻴﺠﻌل ﻤﻨﻬﺎ ﻭﺴﻴﻠﺔ ﻋﺎﻟﻴﺔ ﺍﻟﻔﺎﻋﻠﻴﺔ ﻭﻤﻨﺎﺴﺒﺔ ﺒﺸﻜل ﺨﺎﺹ ﻷﻏﺭﺍﺽ ﺍﻟﺭﻗﺎﺒﺔ‬
‫ﺍﻟﻨﻭﻋﻴﺔ ﻭﺍﻟﺘﺤﻠﻴل‪.‬‬
‫ﺍﻟﻜﻠﻤـﺎﺕ ﺍﻟﺩﺍﻟـﺔ‪ :‬ﺃﻤﻜﺎﻟﻴﻥ‪ ،‬ﺍﻟﺠﺎﺭﺩﻴﻨﺎ )ﺒﻼﺭﺠﻭﻨﻴﻭﻡ(‪ ،‬ﺘﺤﻠﻴل ﺍﻟﻜﺭﻭﻤﺎﺘﻭﻏﺭﺍﻓﻴﺎ ﺍﻟﺴﺎﺌﻠﺔ ﻋﺎﻟﻴﺔ ﺍﻷﺩﺍﺀ ﻭﺍﻟﻤﻜﺸﺎﻑ ﻓﻭﻕ‬
‫ﺍﻟﺒﻨﻔﺴﺠﻲ‪ ،‬ﺸﺭﺍﺏ ﺍﻟﺴﻌﺎل‪ ،‬ﻤﻨﺘﺞ ﻨﺒﺎﺘﻲ‪ ،‬ﺩﺍﻟﺔ )ﻭﺍﺼﻤﺔ( ﻨﺒﺎﺘﻴﺔ‪.‬‬

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‫ﺘﺎﺭﻴﺦ ﺍﺴﺘﻼﻡ ﺍﻟﺒﺤﺙ ‪ 2009/11/22‬ﻭﺘﺎﺭﻴﺦ ﻗﺒﻭﻟﻪ ﻟﻠﻨﺸﺭ ‪.2010/3/14‬‬

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