Acevska et al.: Journal of AOAC International Vol. 95, No.
2, 2012 399
DIETARY SUPPLEMENTS
Development and Validation of a Reversed-Phase HPLC Method
for Determination of Alkaloids from Papaver somniferum L.
(Papaveraceae)
Jelena Acevska and Aneta Dimitrovska
University Ss. Cyril and Methodius, Institute of Applied Chemistry and Pharmaceutical Analysis, Faculty of Pharmacy,
Vodnjanska 17, 1000 Skopje, Republic of Macedonia
Gjoshe Stefkov
University Ss. Cyril and Methodius, Institute of Pharmacognosy, Faculty of Pharmacy, Vodnjanska 17, 1000 Skopje,
Republic of Macedonia
Katerina Brezovska
University Ss. Cyril and Methodius, Institute of Applied Chemistry and Pharmaceutical Analysis, Faculty of Pharmacy,
Vodnjanska 17, 1000 Skopje, Republic of Macedonia
Marija Karapandzova and Svetlana Kulevanova
University Ss. Cyril and Methodius, Institute of Pharmacognosy, Faculty of Pharmacy, Vodnjanska 17, 1000 Skopje,
Republic of Macedonia
An HPLC method for the separation of six target
alkaloids from Papaver somniferum L. (morphine,
codeine, oripavine, thebaine, papaverine, and
noscapine) was developed, optimized, and validated.
The chromatographic behavior of these alkaloids was
investigated using a reversed-phase chromatography
at acidic and alkaline pH. The effects of ion-pairing
agents, pH value of the mobile phase, concentration
of the buffer components, mobile phase organic
modifier, and column temperature were studied.
Regardless of the large differences in their pKa
values, all alkaloids were separated within a close
retention window, and good peak shape was achieved
for each of the six alkaloids. The proposed method
has adequate selectivity, linearity, accuracy, precision,
and reproducibility and is applicable for poppy straw.
T
he opium poppy, Papaver somniferum L. (Papaveraceae),
is one of the oldest medicinal plants. It has been
utilized and cultivated since prehistoric times for the
narcotic and nutritive values of its products (1–3). More than
40 individual alkaloids have been isolated from opium poppy.
Only five of these alkaloids account for virtually all of the
quantitative alkaloid content in opium, including the morphinans
(morphine, codeine, and thebaine), the benzylisoquinoline
(papaverine), and the phthalideisoquinoline (noscapine;
Figure 1). Traces of other alkaloids exist and are compiled in
the following alkaloid classes: aporphines, protoberberines and
tetrahydroprotoberberines, rhoeadines, benzophenanthridines,
and tetrahydroisoquinolines (1). The application of poppy
alkaloids is restricted in some well-defined therapeutic fields.
Analgesics of morphine origin are used mainly to control severe
pain; they also have antidiarrhea and sedative effects. Codeine,
Received March 19, 2011. Accepted by AP May 18, 2011.
Corresponding author’s e-mail: jpetrusevska@yahoo.com
DOI: 10.5740/jaoacint.11-102
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pholcodine, ethyl morphine, and noscapine are used to elevate
antitussive activity. Papaverine is used as a smooth muscle
relaxant (3). Thebaine and oripavine are used as starting material
for production of synthetic and semisynthetic derivatives of the
opiates.
According to the International Narcotics Control Board
of the United Nations (4), the preceding 20 years have seen
continually increasing demand for natural alkaloids (morphine,
codeine, thebaine, and oripavine) obtained from the opium poppy
plant. About 83% of the morphine and 91% of the thebaine
manufactured worldwide are obtained from poppy straw, the cut
and dried upper stalk of the plant, including the crushed capsules.
Literature data for determination of alkaloids from
P. somniferum L. describe analysis of substances and drug
products (5, 6), poppy alkaloids in opium (7), and alkaloids in
plant material (Papaver sp.; 8–11), as well as monitoring the
opiates and their metabolites in biofluids (12, 13).
The numerous patents for creation of Papaver strains with high
content of certain alkaloids (14–17), and extraction and isolation
procedures from opium and plant material (18–25), confirm the
continuous scientific interest and economic significance of this
subject. Many applications of simultaneous analysis of the major
opium alkaloids, using chromatographic (26–34) or electrophoretic
separation techniques (35) have been published. Most of the
chromatographic methods for determination of the alkaloids
comprise HPLC with diode-array detection (7–12, 26, 27), HPLC
with fluorescence detection (28), LC/MS (29–31), GC/MS (32–
34), and HPTLC (9, 10).
HPLC has been proven to be most efficient for separation
and quantification of opium alkaloids. Although some normal
phase chromatography methods have been published, most of the
applications use reversed-phase chromatography with gradient
elution, which is vital due to the large difference in the polarity
of the five major opium alkaloids. The majority of the actual
methods, including the official pharmacopeial method (36) for
assay and impurities in standardized dry extract of raw opium,
demand the use of ion-pairing agents (sodium dodecyl sulfate
or alkyl sulfonates) and an acidic mobile phase on reversed-
phase stationary phases (6, 8, 26). Тhese separation systems
400 Acevska et al.: Journal of AOAC International Vol. 95, No. 2, 2012

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Figure 1. Chemical structure of morphine, codeine, oripavine, thebaine, papaverine, and noscapine.

are not compatible with MS detection due to the nonvolatile
ion-pair reagents. Many of the reported methods are restricted
to the quantification of two to four of the major poppy alkaloids.
Furthermore, there are imposed demands for reduced use of
organic solvents due to their high price and problems regarding
waste disposal.
Because of the excessive interest of alkaloid simultaneous
assaying, especially in the breeding programs of P. somniferum
cultivars, this study aimed to develop and optimize a fast,
inexpensive, convenient, and performable HPLC method,
suitable for accomplishing the large number of analyses needed to
assess the content of the six target alkaloids (morphine, codeine,
oripavine, thebaine, papaverine, and noscapine) in poppy
straw. The chromatographic behavior of the target alkaloids
on a reversed-phase column with bidentate C18-C18 bonding
technology over a wide range of pH values was also studied.
Sample Preparation
Mature poppy capsules and stems (up to 10 cm length) were
collected and trashed to remove the seeds, thus forming a straw.
Proximally, 0.1 g (accurately weighed) of dry, pulverized poppy
straw (mesh 0.45 mm) was extracted with 5 mL methanol by
ultrasonic agitation (60 kHz, 40°C) for 20 min. The extract was
centrifuged (4000 rpm, 5 min), and the supernatant was filtered
and collected. The solid remaining was submitted to a repeated
extraction procedure with another 5 mL methanol. The filtrates
were combined and diluted to 10.0 mL with the same solvent.
After filtration through a 0.45 µm Millipore (Bedford, MA) filter,
the samples were analyzed by HPLC.
Apparatus

Experimental The Agilent 1200 HPLC system (Agilent Technologies GmbH,

Materials
Authentic samples of morphine, codeine, oripavine, thebaine,
noscapine, papaverine (all as free bases), and poppy straw were
obtained from Alkaloid A.D., Skopje (Skopje, R. Macedonia).
Boeblingen, Germany) was equipped with a vacuum degasser
(G1322A Degasser), quaternary pump (G1311A Quat Pump),
autosampler (G1329A ALS), column compartment (G1316A
TCC), and diode array detector (G1315D DAD). ChemStation for
LC 3D software (Wilmington, DE) was used for data handling.

Reagents

Acetonitrile (HPLC grade), methanol (HPLC grade),

trifluoroacetic acid (TFA; for synthesis), and phosphoric acid 85%
(pro-analysis) were purchased from Merck, Darmstadt, Germany.
Triethylamine (TEA; pro-analysis) and sodium heptansulfonate
monohydrate (Puriss. p.a, for ion-pair chromatography) were
purchased from Sigma-Aldrich, Steinheim, Germany. Water
(highly purified) was obtained with a TKA-LAB Reinstwasser
system (Niederelbert, Germany).

Standard Solution of Alkaloids Figure 2. Chromatogram of alkaloid separation.

Alkaloid mixture was prepared from methanolic stock
solutions of 4 mg/mL morphine, 2 mg/mL codeine, 1 mg/mL
thebaine, 1 mg/mL oripavine, 1 mg/mL papaverine, and 2 mg/mL
noscapine, to obtain a concentration of 200 µg/mL morphine;
100 µg/mL codeine and noscapine; and 50 µg/mL thebaine,
papaverine, and oripavine.
Chromatographic conditions: Zorbax Extend C-18
column, 250 × 4.6 mm id, 5 μm particle size; eluents:
methanol and 0.1% TFA in water, pH adjusted with
TEA to 9.6; gradient elution: 0–2 min 45% methanol;
2.1–10 min from 45 to 70% methanol; 10–20 min
from 70 to 85% MOH; flow rate, 1.5 mL/min; injection
volume, 20 μL; column temperature, 30°C; and
detection at 280 nm.
 Acevska et al.: Journal of AOAC International Vol. 95, No. 2, 2012 401

Table 1. System suitability valuesa

Parameter Morphine Oripavine Codeine Papaverine Thebaine Noscapine

Rt 4.6 6.5 7.6 9.9 10.5 13.5
k′ 2.7 4.2 5.1 7.0 7.4 9.8
a 1.47 1.20 1.32 1.06 1.33
As 0.83 0.77 0.73 0.81 0.66 0.82
N 5444 12971 20728 45069 35036 68169
Rs 7.8 5.0 11.6 2.4 13.9

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RSD (Rt) 0.72 0.53 0.29 0.18 0.11 0.09
RSD (AUC) 0.95 0.69 0.63 0.63 0.67 0.51
a
 Rt = Retention time; k′ = capacity factor; α = selectivity; As = symmetry factor; N = number of theoretical plates;
Rs = resolution; RSD (Rt) = RSD of the retention time; RSD (AUC) = RSD of the peak area.

Chromatographic Conditions of morphine and codeine along with accompanying substances,

Preliminary method development was performed with a
250 mm × 4 mm id, 5 µm particle size, stainless-steel Symmetry
C-8 column (Waters Corp., Milford, MA). Final quantification
was performed on a 250 mm × 4.6 mm id, 5 µm particle size,
Zorbax Extend C-18 column (Agilent Technologies). The
eluents were a solution of 0.1% TFA in water, pH adjusted to
9.6 with TEA (solvent A) and methanol (solvent B), both filtered
through 0.45 µm Millipore filters. Elution was performed with
the gradient: 0–2 min 45% solvent B; 2–10 min from 45 to 70%
solvent B; 10–20 min from 70 to 85% solvent B; 20–20.1 min
from 85 to 45% solvent B; 20.1–30 min 45% solvent B. The flow
rate was 1.5 mL/min and the injection volume was 20 µL. The
column temperature was maintained at 30°C, and detection was
carried out at 280 nm.
Validation of the Method
Validation of the method included determination of specificity,
linearity, accuracy, precision, LOD, LOQ, and robustness
according to ICH guidelines (37), taking into account the
recommendations of other appropriate guidelines (38–40).
Results and Discussion
The method recommended by the European Pharmacopoeia
for the quality assessment of the opium dry extract, standardized
(Ph. Eur. 7.0: 01/2008:1839 corrected 6.0), using a 250 × 4 mm id,
5 µm particle size, stainless-steel C-8 column and mobile phase
of sodium heptanesulfonate as an ion-pairing agent in the acidic
mobile phase, provides system suitability criteria for assaying
morphine and codeine, as well as thebaine as an impurity. As
expected, isocratic conditions were not appropriate for the
separation of all six alkaloids because of the large k′ values of
noscapine (>20). For the separation of the target alkaloids, it was
necessary to bring in gradient elution. Literature data show that
such chromatographic conditions can be very sensitive to minor
changes in mobile phase pH, mobile phase organic percentage,
and column temperature (41).
On the other hand, most of the problems that can occur in
the analysis of poppy straw are interferences from the matrix,
lack of resolution of at least two major alkaloids, rapid elution
or broad peak shape of late-eluting alkaloids (26). In addition,
possible strong secondary interactions between acidic silanols
and the amine groups of the alkaloids can cause peak tailing and
poor efficiencies with bonded silica packing materials.
Because of the basic nature of alkaloids, it would be preferable
to analyze these compounds at an alkaline pH, where the amine
group would not be ionized. This is not generally possible
with bonded silica-based columns because of the instability
of the silica matrix at alkaline pH (26, 41). Modified bonded
silica, polystyrene divinylbenzene phases, alumina-based
polybutadiene-coated phases, and porous graphitic carbon are
stationary phases that are stable at alkaline pH (27, 42).
Choice of Stationary and Mobile Phases
Ion-pairing agents are not necessary for the separation of the
alkaloids at basic pH, unlike the situation at acidic pH, where
the use of an anionic ion-pairing agents is required to avoid peak
tailing and achieve a reasonable separation. In order to avoid use
of ion-pairing agents, we searched for alternative mobile phase.
Some of the mobile phase buffer options for high pH include
TEA, pyrrolidine, glycine, borate, and ammonium hydroxide in
combination with TFA (8–11, 26). The presence of TFA in the
mobile phase changes the separation mechanism on a reversed-
phase system because it is not a classical ion-pair reagent (43, 44).
We decided to use an eluent system of acetonitrile–water–TEA–
Figure 3. Influence of mobile phase pH value on
chromatographic behavior of morphine, codeine,
oripavine, thebaine, papaverine, and noscapine,
estimated through capacity factor (k′) values.
402 Acevska et al.: Journal of AOAC International Vol. 95, No. 2, 2012

Table 2. Effect on different organic modifier in the mobile phase on capacity factor (k′) and critical resolution
(Rs) of the target alkaloids

Acetonitrile and TFA/TEA pH 9.6 Methanol and TFA/TEA pH 9.6

Chromatographic (0–2 min 20%; 2–5 min 28%; 5.1–10 min 40%; (0–2 min 45%; 2.1–10 min from 45 to 70%;
condition 10.1–0 min 55% acetonitrile) 10–20 min from 70 to 85% methanol)
Parameter Rt k′ Rs Rt k′ Rs
Morphine 4.4 2.5 / 4.7 2.8 /
Oripavine 6.9 4.5 12.1 6.4 4.1 7.4
Codeine 7.3 4.8 2.3 7.4 4.9 5.2

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Papaverine 10.5 7.4 18.6 9.4 6.5 11.3
Thebaine 11.6 8.3 4.8 9.8 6.8 2.4
Noscapine 16.0 11.8 18.2 12.6 9.1 14.4

TFA mixtures, in a wide range of mobile phase pH to separate and noscapine was investigated on different mobile phase pH
the alkaloids from accompanying substances on a reversed-phase values (3.2, 4.0, 5.0, 7.0, 8.5, 9.0, 9.6, 10.0, and 10.5), and
column with bidentate C18-C18 bonding technology that prevents was estimated through capacity factor (k′) values, as shown in
interactions between noncharged basic compounds with the Figure 3.
underlying silica at high pH (42). We also investigated the effect Although all alkaloids were eluted at an acidic pH range (<5.0),
of mobile phase pH, mobile phase organic modifier, concentration k′ values of morphine, codeine, and oripavine were unsatisfactory
of TEA/TFA in the buffer, and the column temperature. at pH <2. At pH 4.0 and 5.0 a significant asymmetry of noscapine
The separation of all six alkaloids was obtained with optimized
peak was evident. Additionally, at acidic ambience, a peak was
gradient elution with methanol and TEA/TFA, pH adjusted to 9.6.
eluted with the column dead volume possessing absorption
A typical chromatogram is shown in Figure 2, with all compounds
spectra similar to that of the morphinan alkaloids. This is
being fully resolved and eluted within 14 min. Retention times
were: morphine, 4.6; oripavine, 6.5; codeine, 7.6; papaverine, probably a consequence of the occurrence of different ionizable
9.9; thebaine, 10.5; and noscapine, 13.5, with resolution of at forms of some of the alkaloids. This thesis should be elucidated
least 2.4 (Table 1). with further MS analysis; the peak did not occur at basic pH
(>7.0). Neutral pH was not suitable for separation of codeine
Effect of Mobile Phase pH on Retention and Peak Shape and oripavine, as well as papaverine and thebain, due to low
resolutions. At pH 8.5, an inversion elution occurred within the
The most important part of the method development was the pairs of peaks for oripavine and codeine, as well as for thebain
choice of pH value of the mobile phase, because the retention and papaverine. In the basic pH range (8.5–10.5), k′ values for
of the alkaloids varied as a function of pH, with the ionized all peaks except noscapine, are between the target range of 2
form being the least retained. The effect of pH on retention of to 10 (46). At pH 9.0 oripavine and codeine are eluted as one
the compounds appeared to be directly related to the degree peak. At pH 9.6, all peaks are eluted, and it may be possible to
of ionization of the individual compounds, complying with optimize the chromatographic conditions in order to improve the
a reversed-phase mechanism of separation. To understand critical resolution between oripavine and codeine, and to obtain
the effect of pH on retention it was necessary to consider the a satisfactory k′ value for noscapine. At extreme basic conditions
ionization constants (pKa) for each of the ionizable groups on
the analytes. Morphine, codeine, oripavine, thebaine, papaverine,
and noscapine (chemical structures shown in Figure 1) have a
number of different ionizable groups, but there are no precise
values for ionization constants for these compounds in the
literature (45). All these alkaloids contain a tertiary amine with a
pKa value >8.2. Between pH 6 and 10, retention of the alkaloids
is increased because of the loss of positive charge from the amine
group. For the compounds with an ionizable phenolic hydroxyl
group (pKa about 9.5) there was a marked reduction in retention
above pH 9.5 as the ionization of this group increased. The
retention of alkaloids (noscapine and papaverine) having acidic
groups (pKa about 6) was increased at pH <5. This confirms that
the reduction in ionization of these acidic groups has a direct
effect on retention. Additional complexity occurs because of the
amphoteric properties of morphine, oripavine, and codeine. Figure 4. Influence of column temperature on
In order to choose the pH value of the mobile phase, we profiled retention of morphine, codeine, oripavine, thebaine,
papaverine, and noscapine, estimated through
the pH of components in the mixture. The chromatographic
capacity factor (k′) values.
behavior of morphine, codeine, oripavine, thebaine, papaverine,
 Acevska et al.: Journal of AOAC International Vol. 95, No. 2, 2012 403

Table 3. Results obtained from testing different parameters during validation of the analytical method
Validation parameter Morphine Oripavine Codeine Papaverine Thebaine Noscapine
Specificity Peak purity ratio 0.001 0.003 0.037 0.017 0.005 0.059
Linearity Conc. range, µg/mL 50–300 12.5–75 25–150 12.5–75 12.5–75 25–150
R2 0.9993 0.9990 0.9990 0.9991 0.9990 0.9991
Intercept –16.598 –5.945 –0.065 –0.903 –9.476 –3.053
Slope 3.711 17.258 3.763 15.694 19.156 5.196
LOD, µg/mL 1.8 0.3 0.6 0.3 0.2 0.5

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LOQ, µg/mL 5.4 0.9 1.8 0.8 0.6 1.6
Precision (RSD), %
Analyst 1 (n = 6) 0.95 0.69 0.63 0.63 0.67 0.51
Analyst 2 (n = 6) 0.25 0.23 0.22 0.20 0.19 0.14
Reproducibility (RSD), %
Laboratory 2 0.44 0.46 0.38 1.11 1.37 0.31

(pH 10.0 and 10.5), the k′ value for morphine is again small (<2)
and oripavine elutes as doublet.
Baseline separation of all alkaloids was obtained with
optimized gradient elution at pH 9.6. A step-by-step gradient
led to a chromatogram with all compounds being fully resolved
and eluted within 20 min retention time. The order of elution
was morphine, oripavine, codeine, thebaine, papaverine, and
noscapine, with morphine well-separated from the solvent front.
Effect on Different Organic Modifier in the Mobile Phase
In order to reduce the retention times and provide the best
peak shape, the effect of different organic modifiers (acetonitrile
and methanol) was investigated. Using similar chromatographic
conditions, acetonitrile reduced the retention of the compounds
compared to methanol, but at the same time, it diminished the
values of the critical resolution (Table 2). Further opportunities
for optimization of the critical resolutions have arisen by using
methanol for the gradient, as an eluent with weaker elution power
than acetonitrile. Because methanol was used as solvent for the
sample preparation (extraction of the alkaloids from poppy straw),
it facilitated better peak shape. Methanol has a lower price and less
toxicity than acetonitrile (47), thus gaining an additional advantage
over acetonitrile and influencing the final decision to include it
as eluent of the proposed method. Optimization of the gradient
resulted in satisfactory separation of all compounds (Figure 2).
Concentration of TFA was tested in the range of 0.05 to 0.3%
(v/v), and concentration of TEA in the range of 0.1 to 0.6%
(v/v). The pH of the mobile phase was maintained at 9.6. Results
showed that the concentration of TFA and TEA had insignificant
influence on the retention of the components. Decreasing the
concentration of both reagents resulted in decreased baseline
noise (increased sensitivity) and slightly increased the column
efficacy (insignificant increased number of theoretical plates
and improved peak symmetry). From the obtained results,
taking into account proper maintenance of the column, we chose
concentrations of 0.1% (v/v) TFA and 0.2% (v/v) TEA in the
mobile phase buffer.
Effect of Temperature
An approximately linear reduction in k′ values was observed
over the range of studied temperatures (25, 30, 35, and 40°C), and
a small increment of the chromatographic efficiency was obtained
for all the alkaloids (Figure 4). The temperature increment
triggered better solubility of the solutes in the mobile phase,
facilitating larger mass transfer of the solute into the stationary
phase (48). The reduced retention times at higher temperatures
are practical for performing faster run times, while retaining
sufficient selectivity and resolving individual compounds. Since
the difference in chromatographic efficacy was not significant,
the column temperature chosen was 30°C.

Concentration of Buffer Components Validation of the Method

Concentrations of both buffer components (TEA and TFA) A typical chromatogram is shown in Figure 2. The results
were adjusted to obtain adequate sensitivity of the method. from testing the system suitability of the method are given in

Table 4. Results obtained from testing the accuracy of the method (morphine)

Concn, % Present in sample solution, µg Added, µg Recovery, % (n = 3) RSD, % (n = 3) P, 95% confidence interval
25 153.02 40.98 99.50 0.43 99.50 ± 1.06%
50 153.02 81.96 99.08 0.17 99.08 ± 0.42%
100 153.02 163.93 97.34 0.33 97.34 ± 0.80%
404 Acevska et al.: Journal of AOAC International Vol. 95, No. 2, 2012

Table 5. Results of tests for robustness of the method

Morphine Oripavine Codeine Papaverine Thebaine Noscapine Resolution (critical)

Parameter (k′) (k′) (k′) (k′) (k′) (k′) (Rs)
Concentration of methanol (in initial isocratic step of the gradient)
40% (v/v) 3.74 5.24 6.05 7.65 8.00 10.05 2.33
45% (v/v) 2.73 4.24 5.14 7.01 7.40 9.77 2.37
50% (v/v) 1.60 2.54 3.41 5.37 5.77 8.66 1.92
Column temperature, ºС

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25 2.87 4.42 5.31 7.20 7.58 10.00 2.00
30 2.73 4.24 5.14 7.01 7.40 9.77 2.37
35 2.56 4.03 4.93 6.82 7.21 9.57 2.40
Mobile phase, рН
9.5 2.73 4.25 5.15 7.01 7.39 9.78 1.64
9.6 2.72 4.23 5.12 6.99 7.38 9.77 2.37
9.7 2.77 4.29 5.18 7.03 7.44 9.83 1.81

Table 1. The column efficiency was determined by the number
of theoretical plates (N). The obtained values for all analytes (the
smallest value for morphine, N >5000) indicates good separation
efficiency of the applied column. Capacity factor values for all
peaks were between 2 and 10. All peaks had good shape, with
symmetry factor values close to 1.
Figure 2 shows that, under the proposed chromatographic
conditions, all alkaloids were completely separated from
each other (critical pair of analytes were papaverine and
thebain, selectivity α = 1.06, resolution Rs = 2.4). Purity of
the peaks corresponding to the alkaloids was determined using
ChemStation for LC 3D software for data handling. The purity
(similarity/threshold) ratio was within the calculated threshold
limit (1.000) for all the alkaloids (Table 3). The method was
found to be specific.
The repeatability of the method was determined from six
injections of the standard solution. The obtained values for the
RSD (n = 6) of the retention times and peak areas (Table 1)
were <2%, which confirmed that the method was precise. The
intermediate precision of the method was determined by two
analysts who tested the sample solution under independent
conditions; there was no significant difference in precision
(Table 3). The reproducibility of the method was assessed
through conduction of the proposed analytical procedure in
anther independent laboratory in order to ensure easy method
transfer. There was no significant difference in obtained values
for the RSD (n = 6) of the peak areas (Table 3) between the two
laboratories, which confirmed that the method was reproducible.
Over the concentration range selected, peak area was linearly
dependent on concentrations for all the compounds. The results
obtained from the regression analysis of the peak area (y) versus
concentration (x) data (Table 3) indicated that the method was
linear (correlation coefficients were >0.999) in the concentration
range from 25 to 150% (50–300 µg/mL for morphine,
25–150 µg/mL for codeine and noscapine, and 12.5–75 µg/mL
for thebaine, oripavine, and papaverine). The obtained regression
2
equations were: y = 3.711x – 16.598, R = 0.9993 for morphine;
2
y = 3.763x – 0.065, R = 0.9990 for codeine; y = 17.258x – 5.945,
2 2
R = 0.9990 for oripavine; y = 19.156x – 9.476, R = 0.9990 for
thebaine; y = 15.694x – 0.903, R2 = 0.9991 for papaverine; and
2
y = 5.196x – 3.053, R = 0.9991 for noscapine.
The LOD and LOQ were determined from the residual SD
of the regression line (σ) and the slope (S) in the concentration
range from 1 to 25%. The LOD values (3.3 σ/S) were
1.8 µg/mL for morphine, 0.3 µg/mL for oripavine, 0.6 µg/mL
for codeine, 0.3 µg/mL for papaverine, 0.2 µg/mL for thebaine,
and 0.5 µg/mL for noscapine. The LOQ values (10 σ/S) were
5.4 µg/mL for morphine, 0.9 µg/mL for oripavine, 1.8 µg/mL for
codeine, 0.8 µg/mL for papaverine, 0.6 µg/mL for thebaine, and
1.6 µg/mL for noscapine (Table 3).
Recovery values (Table 4) obtained from determination of
morphine using the methods of standard additions confirmed that
the method was accurate in the range from 25 to 100% of the
working concentration. The spiking of the sample was performed
by adding the standard of morphine to the poppy straw at the
beginning of the extraction procedure. Mean recoveries for
morphine were 99.5% (P95% = ±1.1%) at 25% of the working
concentration, 99.1% (P95% = ±0.4%) at 50% of the working
concentration, and 97.3% (P95% = ±0.8%) at 100% of the working
concentration.
The results from the determination of the robustness of
the method indicated that small but deliberate changes in
the analytical conditions, such as temperature of the column
(±5°C), pH value of the mobile phase (±0.1), and change in the
concentration of the organic modifier [±5% (v/v) methanol] in
the initial isocratic step of the gradient, did not affect the critical
resolution between the peaks (Table 5), and that the method can
be applied in routine work.
Conclusions
A convenient and performable HPLC method was developed
and optimized for determination of morphine, codeine, thebaine,
oripavine, papaverine, and noscapine in poppy straw. Results
from validation of the method show satisfactory specificity,
linearity, accuracy, precision, and reproducibility. The indicated
robustness of the method guarantees unambiguous application of
the method in routine work. The proposed HPLC method is fast
 Acevska et al.: Journal of AOAC International Vol. 95, No. 2, 2012
and suitable for assessing the content of the target alkaloids in
poppy straw.
Acknowledgments
A part of this study was financially supported by ALKALOID
AD Skopje, Republic of Macedonia.
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