PROCESSING AND PRODUCTS
Method for the determination of chromium in feed matrix by HPLC
Balakrishnan Umesh,∗,1 Rajendra Moorthy Rajendran,∗ and Muthu Tamizh Manoharan†
∗
Research and Development, Kemin Industries South Asia Pvt. Ltd., India, The Trapezium, 39, Nelson
Manickam Road, Chennai, Tamil Nadu, India 600029; and † Interdisciplinary School of Indian System of
Medicine, SRM University, Kattankulathur 603203, Tamil Nadu, India
ABSTRACT An improved method for the chromatographic separation and determination of chromium (III)
and (VI) [Cr(III) and Cr(VI)] in mineral mixtures
and feed samples has been developed. The method
uses precolumn derivatization using ammonium pyrrolidinedithiocarbamate (APD) followed by reversedphase liquid chromatography to separate the chromium
ions. Both Cr(III) and Cr(VI) species are chelated with
ammonium pyrrolidinedithiocarbamate prior to separation by mixing with acetonitrile and 0.5 mmol acetate
buffer (pH 4.5). Optimum chromatographic separations
were obtained with a polymer-based reversed-phase column (Kinetex, 5 μ, 250 × 4.5 mm, Phenomenex, Torrance, CA) and a mobile phase containing acetonitrile
and water (7:3). Both Cr(III) and Cr(VI) ion concentrations were directly determined from the corresponding
areas in the chromatogram. The effect of analytical pa-
rameters, including pH, concentration of ligand, incubation temperature, and mobile phase, was optimized for
both chromium complexes. The range of the procedure
was found to be linear for Cr(III) and Cr(VI) concentrations between 0.125 and 4 μg/mL (r2 = 0.9926) and
0.1 and 3.0 μg/mL (r2 = 0.9983), respectively. Precision
was evaluated by replicate analysis in which the percentage relative standard deviation values for chromium
complex were found to be below 4.0. The recoveries
obtained (85–115%) for both Cr(III) and Cr(VI) complexes indicated the accuracy of the developed method.
The degradation products, as well as the excipients,
were well resolved from the chromium complex peak in
the chromatogram. Finally, the new method proved to
be suitable for routine analysis of Cr(III) and Cr(VI)
species in raw materials, mineral mixtures, and feed
samples.
Key words: total chromium, HPLC validation, mineral mixtures, hexavalent chromium, chromium speciation
2015 Poultry Science 94:2805–2815
http://dx.doi.org/10.3382/ps/pev238
INTRODUCTION
Some transition metals can induce adverse actions
in a biological system with different toxicity expressions depending on their oxidation state. Chromium
is considered to be a bio-element in the trivalent form
(Anderson, 1995) or to have mutagenic properties in the
hexavalent form (Katz and Salem, 1993). Chromium
is an essential mineral in animal nutrition and is used
in feed supplements for various applications, such as
glucose homeostasis, growth performance, and antidepressant effects (Anderson and Kozlovsky, 1985; Mazzer
et al., 2007; Rajalekshmi et al., 2014). The hexavalent
form of chromium is highly toxic and, thus, is permissible at a level of less than 0.1 mg/L in drinking water,
according to the United States Environmental Protection Agency (US EPA). Hence, it is essential to quantify
both ions at trace levels in all products.
C 2015 Poultry Science Association Inc.
Received September 17, 2014.
Accepted July 9, 2015.
1
Corresponding author: [email protected]
Chromium propionate and chromium picolinate are
the two organic forms of chromium permitted by the
U.S. Food and Drug Administration (FDA) for addition to swine diets up to 0.2 mg/kg, whereas in cattle feed chromium propionate is the only approved
chromium source at a dosage of 0.5 mg/kg by the
FDA. Hence, most chromium-based feed supplements
contain low levels of the Cr(III) ion, along with stabilizers, fillers, silica, and other ingredients. There have
been a large number of reports over the past few
years with regard to specific analytical methods for
the speciation of chromium ions individually in water (Ashraf et al., 2006). At present, atomic absorption spectroscopy (Sperling et al., 1992), inductively
coupled plasma (ICP) and ion chromatography (Inoue
et al., 1995) are widely used for solid matrix analysis.
Wang (2010) developed a method for simultaneously
quantifying chromium ions using inductively coupled
plasma-mass spectrometry (ICP-MS) in urine from
chromate workers. However, high-performance liquid
chromatography (HPLC) is one of the most accurate,
cost-effective, and widely used techniques in the analytical lab. By developing an HPLC method to quantify
2805
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UMESH ET AL.
Cr(III) and Cr(VI) ions simultaneously in solid matrices, the time and cost of the analysis will be reduced.
Several reports have been published on using
reversed-phase high-performance liquid chromatography (RP-HPLC) techniques for estimating chromium
ion concentration in aqueous media. The majority of
these methods have used ion exchange resins for preconcentration and co-precipitation for quantification.
Kaur and Malik (2009) proposed a simultaneous determination method of chromium compounds by RPHPLC using morpholine-4-carbodithioate ligands with
UV detection. The method has a broad dynamic range
but is rather time consuming. Analysis of Cr(III) and
Cr(VI) by co-precipitation using Fe(III) oxide and
extracted with ammonium pyrrolidine thiocarbamate
(Mullins, 1984). Several reports have been published
on chelation extraction procedures using sodium pyrrolidinethiocarbamate ligands for simultaneous determination of chromium species in water (Tande et al., 1980;
Subramanian, 1988). The RP-HPLC method was validated for the determination of chromium ions using
ammonium pyrrolidine (APD) ligands in a photodiode
array (PDA) detector in water samples (Hossain et al.,
2005). However, none of these methods were found to be
optimized for the analysis of chromium in solid matrices like mineral mixtures or feed supplements for better
recoveries. This is due primarily to the interference of
other ions and tedious experimental methods for extraction.
Thus, a precolumn derivatization procedure using
ammonium pyrrolidinethiocarbamate ligands for selective chromium estimation was attempted with an approach modified from the reported procedure (Tande
et al., 1980). To optimize the HPLC condition, both
complexes were individually isolated and the structures were confirmed using Fourier transform infrared
spectroscopy (FTIR), electron paramagnetic resonance
(EPR), and liquid chromatography–mass spectrometry (LC-MS). The complexes of Cr(III)-APD and
Cr(VI)-POS have different types of polar attraction
with organic solvents, but they have similar UV absorption characteristics. Therefore, Cr(VI)-POS and
Cr(III)-APD complexes can be separated from their
mixtures with RP-HPLC using a suitable composition
of organic and aqueous mobile phases.
In this paper, a comprehensive study of the chelation
mechanism of chromium ions, their structural characterization, and their chromatographic separation using
RP-HPLC is described. The parameters of the chelation
process, including time, temperature, pH, buffer, and
substrate concentration, are discussed. For quantification using HPLC, parameters such as linearity, specificity, precision, accuracy, and robustness are validated
in accordance with the International Conference on
Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use (ICH) guidelines. The developed method was applied to analyze total chromium and hexavalent chromium in several solid
matrices, including feed samples and mineral mixtures.
MATERIALS AND METHODS
Reagents
All organic solvents were of analytical grade available on the market. Acetonitrile and acetic acid were
HPLC grade from Sigma-Aldrich (Bangalore, India).
Deionized water was of HPLC grade quality filtered
through a 0.2 micron filter from Fisher Scientific. All
HPLC solvents were degassed with an ultrasonic bath
prior to use. Chromium chloride hexahydrate (99.9%),
potassium dichromate (99.9%), and ammonium pyrrolidinedithiocarbamate (APD, 99.8%) were obtained
from Sigma-Aldrich (Bangalore, India). Sodium acetate
trihydrate (Merck, India) was of laboratory grade quality and used without further purification. Mineral mixture products containing chromium (ChromflexTM C,
KemTRACETM Chromium) were obtained from Kemin
Industries South Asia Pvt. Ltd. (Chennai, India). The
feed sample tested was a broiler finisher feed, formulated with maize, soya, wheat bran, silica, dicalcium phosphate (DCP), and minerals, including copper,
manganese, zinc, cobalt, and iron.
Physical Measurements
The Shimadzu Prominence HPLC unit consisted of
an LC20AC solvent delivery pump and an SPD-20A UV
photodiode array detector, interfaced with LCsolution
Software (ver 1.25). A sample volume of 20 μL was injected into the injection valve. A Phenomenex Kinetex
C18 reversed-phase column of 4.6 × 250 mm filled with
C18 material with 5 μm packing was used for analysis.
The column temperature was maintained at 40◦ C. A
Mettler Toledo pH meter (Model S220) was used in the
study for buffer preparation. The molecular weight of
both chromium chelate complexes was analyzed using
an Agilent 1100 series LC-MS system equipped with
a dual spray electron ionization system. The stretching and bending vibrations of both complexes was analyzed using a Perkin Elmer 65 FTIR series instrument
using KBr discs. Proton nuclear magnetic resonance
(1 H-NMR) analysis of complexes were recorded using
a 500 MHz Bruker Avance AVIII NMR Spectrometer,
using d6 -DMSO as a solvent and TMS as an internal
standard. Powder EPR spectra were recorded using a
JEOL JES-FA200 operating at an X-band frequency
(8.75–9.65 GHz) at room temperature. The magnetic
moments of these complexes were recorded at room
temperature using an Auto magnetic susceptibility balance (MSB) (Sherwood Scientific).
Standards/Procedure
All standards and solutions were prepared using double distilled water filtered through 0.2 μ filter. APD
reagent solution was prepared by dissolving 0.3 g in
100 mL water. A standard solution of 0.5 μg/mL of
Cr(III) was prepared by diluting the stock solution
VALIDATION PARAMETERS
of CrCl3. 6H2 O (10.5 mg in 50.0 mL) at 40 μg/mL
of Cr(III) in deionized water. Cr(VI) stock solution
was prepared by dissolving potassium dichromate (25.5
mg in 50.0 mL) in distilled water to get a solution of
180.3 μg/mL of Cr(VI). A standard solution of 0.1
μg/mL Cr(VI) was prepared from Cr(VI) stock solution in distilled water. Calibration standards containing chromium species at different concentrations were
prepared approximately 30 min before analysis by appropriate dilution of the Cr(III) and Cr(VI) stock solutions in glass standard flasks using distilled water and
were kept at ambient temperature.
HPLC Assay of Cr Complex
The metal complexation was achieved in situ for both
chromium ions. The required volume of Cr stock solution was pipetted into a 10.0 mL flask. To this were
added APD reagent solution (0.3%, 3.0 mL) and acetate buffer (3.0 mL) solution of pH 4.5. The flask was
kept for incubation at 57◦ C for 15–20 min (Hossain
et al., 2005). The solution turned turbid, which indicates chelation of the metal complex formation. The
amount of precipitate formed is directly proportional
to the concentration of chromium ions. The flask was
allowed to cool and the precipitate was dissolved using acetonitrile and made up to volume using the same
solvent in a standard 10.0 mL flask. The solution was
directly injected in HPLC.
Analysis in Solid Matrices
Cr analysis in chromium propionate complex. A
chromium propionate-APD complex solution was prepared by adding 25 mg chromium propionate to a
100.0 mL volumetric flask with 20.0 mL of water. To
this was added 1.0 mL 50% concentrated HCl solution,
along with 5.0 mL water, and the mixture was heated
for 10 min at 110 ± 10◦ C. The stock solution was allowed to cool and then made up to 100.0 mL using distilled water. Then 250 μL stock solution was diluted to
10.0 mL in a volumetric flask and chelation with APD
was prepared as described previously.
Cr analysis in mineral mixtures. A mineral mixture sample (500 mg) was weighed in a silica crucible.
Three mL of 50% concentrated HCl solution was added,
and the sample was digested at 100◦ C for 10 min on a
hot plate. The sample solution was filtered and diluted
to 50.0 mL using distilled water. Then 200 μL of solution was diluted to 10.0 mL in a volumetric flask and
chelation was completed as described earlier.
Cr analysis in feed samples. Feed sample (2g)
was spiked with 1.0 mL of 20.0 μg/mL chromium standard solution and extracted using a 1:1 methanol:water
(20.0 mL) system at 50◦ C, 150 rpm in a shaking incubator. Then the material was filtered, and 2.0 mL filtrate
was taken for analysis in a 10.0 mL volumetric flask and
derivatized with APD as described earlier.
2807
Method Validation
The method was validated according to the ICH
guidelines through the determination of range, linearity, precision, specificity, accuracy, robustness, limit of
detection (LOD), and limit of quantitation (LOQ).
Linearity and range. The purpose of this part of
the study was to establish the linearity of the proposed
method for Cr(III) and Cr(VI). Six separate series of
solutions of Cr(III) (0.125–4 μg/mL) and Cr(VI) (0.10–
3.5 μg/mL) were prepared from the stock solutions and
analyzed (n = 6). The calibration curve was obtained
by plotting the peak area to the concentration of ions,
and least squares regression analysis was performed on
the obtained data.
Precision. The precision of a method expresses the
closeness of agreement between a series of measurements from the same homogeneous sample. A known
concentration of Cr(III) and Cr(VI) ions was prepared,
and both repeatability and reproducibility were evaluated. The repeatability of the method was checked
by injecting 0.5 μg/mL solution of Cr(III) and Cr(VI)
standards (n = 6) into HPLC. The variability of the
method was studied by analyzing the solution 9 times
on the same day (intraday precision) and 6 times on 3
different days (interday precision).
Specificity. To check the specificity of the proposed
method, a standard mixture of Cr(III) and Cr(VI)
was prepared with excipients like silica, calcium carbonate, organic acids, and glycol that are commonly
found in chromium propionate preparations. A comparison of excipient mixture chromatograms with the chromatograms of the standard solution was made along
with the percentage recovery of both analytes.
Accuracy. The accuracy of an analytical method
is the closeness of test results obtained by developed
method to the true value. This approach was based
on the percentage relative error and mean percentage recovery of spiked Cr ions in chromium mixtures.
A known concentration (0.8–1.2 μg/mL) of Cr ion
was spiked with 1.0 μg/mL organic chromium chelate,
and the total recovery was calculated. For Cr(III), the
area corresponding to total Cr was measured in triplicate and percentage recovery was back-calculated. For
Cr(VI), percentage recovery was cross verified using
other analytical methods (ion chromatography) owing
to lower concentrations in feed raw materials.
Limit of quantitation (LOQ) and limit of detection (LOD). The LOQ and LOD parameters were determined on the basis of the signal-to-noise ratio of the
analytes in the chromatogram. The repeatability of the
Cr(III)-APD and Cr(VI)-POS complexes at a particular concentration should be less than 5% for LOQ and
less than 10% for LOD.
Robustness. The robustness of an analytical procedure refers to its capability to remain unaffected by
small and deliberate variations in method parameters.
The robustness of the method was studied by changing the composition of the organic phase by ±5%, pH
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UMESH ET AL.
by ±0.2, and using different C18 columns. Also, variation in the incubation temperature (±10%) and the
concentration of ligands (±0.1%) were evaluated.
RESUTS AND DISCUSSION
Characterization of Chromium Complex
The reaction between chromium salts and APD and
its application to chromium detection in an aqueous
medium have been extensively studied (Schemes 1 and
2). However, the mechanism of complex formation and
its geometry are not well understood. The objective of
the present work was in part to study the complex reaction between chromium ions and APD, thereby optimizing the experimental conditions for maximum detection
and reproducibility in HPLC.
Electronic spectra. The electronic spectra for the
Cr(III)-APD complex recorded in water gave 4 bands
at 240, 254, 317, and 664 nm. The low-intensity band
observed at 664 nm could be attributed to a forbidden
d-d transition. The ligand-to-metal charge transition
(LMCT) band at 254 and 317 nm can be attributed to a
S→Cr charge transfer. The proposed structure was further confirmed by ultraviolet-visible (UV-Vis) spectral
analysis. The high shift in the λmax in the UV region
is a good indication for complex formation.
In the case of dichromate, chromium, which is in
the hexavalent state, is reduced to the trivalent state
by an APDC ligand to form a dithioperoxycarbamate
complex (bis[N,N-pyrrolidine(dithioperoxycarbamateS,S )] [N,N-pyrrolidine(dithioperoxycarbamato-O,S)]
chromium(III)), shown in Scheme 2 (Hossain et al.,
2005). Peaks corresponding to Cr(VI) at 352 and
441 nm were shifted to longer wavelengths (497 and
636 nm), indicating the formation of a trivalent
chromium complex. Also, a peak at 254 and 285 nm
confirms the LMCT complex (Figure 1). The assignment of spectral bands and structural geometry for
both complexes were made based on earlier reports
(Hossain et al., 2005; Setiyanto et al., 2006).
spectra. The IR spectrum of a ligand and metal
complex is shown in Figure 2. The stretching vibration of the mercapto group (SH) of ligands is highly
polarizable and tends to give a very weak signal at
2,710 cm−1 . The disappearance of this band in the spectra of both chromium complexes confirmed the ligand
complex formation. The deprotonation of the SH group
in chelation is confirmed by a blue shift of the υ (CS) band stretching from 835 cm−1 to 825 cm−1 . Significant change in the wavelength to a higher frequency has
been observed upon complexation with a metal ion for
other bands stretching in the fingerprint region. Pyrrolidine vibration bands at 1,409 cm−1 and 1,048 cm−1 of
chromium complexes (Figure 2a, b) shifted to a higher
Scheme 1. Reaction of chromium chloride and ammonium pyrrolidinedithiocarbamate.
Scheme 2. Reaction of potassium dichromate and ammonium pyrrolidinedithiocarbamate.
VALIDATION PARAMETERS
Figure 1. UV-visible spectra of two chromium complexes and ligand for maximum absorbance determination.
frequency, and a change in shape was observed indicating the chelation of this group with the metal ion. There
is a small shift in the bands of Cr(III)-APD and Cr(VI)POS complexes in the spectra, indicating the formation
of a similar coordination complex. The disappearance
of the peak at 890 cm−1 corresponds to Cr-O of dichromate (Figure 2c) and confirms the reduction of Cr(VI)
to Cr(III) for the Cr-POS reaction (Hope et al., 1977).
1
H NMR spectra. The 1 H NMR spectra of the complexes show resonances that are clearly broadened and
shifted by the paramagnetic chromium (III) metal center. The magnetic moments of these complexes were
recorded at room temperature using an Auto-MSB. The
μeff value of chromium (III) complexes ranges between
3.5 and 3.8 B.M. This further supports the idea that
the complexes are paramagnetic corresponding to 3 unpaired electrons, which supports the trivalent state of
chromium. Powder EPR spectra were recorded using
2809
a JEOL-JES-FA200 operating at nX-band frequency
(8.75–9.65 GHz) at room temperature. EPR analysis
was studied to evaluate the geometry of the metal complex shown in Figure 3b. The metal complex was synthesized and isolated in aqueous medium based on the
reported procedure (Tande et al., 1980). For an octahedral d3 electron configuration of chromium (III), the
ground-state electron term is 4 A2g . The g-values were
calculated using the expression g = 2.0023(1–4λ/10
Dq), where λ is the spin orbit coupling constant for
the metal ion. Typical experimental g-values for Cr(III)
compounds having a 6-coordinated octahedral geometry range from 1.95 to 1.98 (Sulekh et al., 2001).
LC-MS analysis. The mass spectra for both
chromium complexes were studied in electrospray ionization mass spectrometry (ESI-MS) analysis. The
molecular weight of the Cr(III)-APD complex from the
mass spectra was found to be m/z 493.80 (Figure 4a),
which confirmed the proposed structure (Scheme 1).
Also, there was another peak at 562.05, indicating
the presence of two chloride ions as a secondary valence in the coordination sphere, which was confirmed
by M+2 isotopic chloride ion peaks in the spectra,
whereas in the case of Cr(VI)-POS, the peroxo complex formation was confirmed from the m/z peak at
506.95 (Figure 4b). The increase in molecular weight for
Cr(VI)-POS confirms the oxide bond coordination in
the pyrrolidine(dithioperoxycarbamato-O,S) chromium
(III) complex. The mass spectral results match the proposed structure in Figure 4, and the results are tabulated in Table 1.
Method Development and Optimization
Optimization of the HPLC method is necessary
for detection and reproducibility at lower levels. This
method is based on precolumn derivatization using
Figure 2. FTIR spectra of a) Cr(III)-APD, b) Cr(VI)-POS, c) K2 Cr2 O7 , and d) APD.
2810
UMESH ET AL.
Figure 3. (a) 1 H-NMR, (b) EPR electronic spectra of Cr(III)-APD, and (c) Cr(VI) complexes.
APD ligands. Hence, both the HPLC method and sample preparation are critical for better reproducibility.
The complexation reaction of Cr(III) and Cr(VI)
with APD are highly dependent on pH. The effect of pH
on complex formation is shown in Figure 5, where the
difference in response for both ions at different pH conditions was observed; it was mainly due to the rate of
dissociation and complex formation of ions with APD.
In the case of Cr(VI), depending on the pH of the solution, it can exist as either CrO4 2− or Cr2 O7 2− . At a
low pH (4–5), only the CrO4 2− ion exists, and it reacts
with water to form hydrogen chromate (HCrO4− ) and
then forms a complex with APD (Scheme 2). In the
case of the positively charged Cr(III) ion, complexation
was high at acidic pH 4.5 and attained saturation at
pH 4.75 (Hossain et al., 2005).
Optimization of the HPLC method was completed
by varying the mobile phase and analytical column to
obtain maximum separation and well-resolved symmetrical peaks. Satisfactory separation and well-resolved
symmetrical peaks were obtained with the mobile phase
containing acetonitrile and water (70:30 v/v) using a
Kinetex C18, 5 μ column. Other parameters, such as
incubation time, oven temperature, and flow rate, were
also studied; the optimized parameters are listed in
Table 2.
System suitability. The suitability of the HPLC instrument is very critical for Cr analysis. The suitability
test parameters are tabulated in Table 3. In the aforementioned HPLC conditions, the elution of the Cr(III)
and Cr(VI) complexes was obtained at a very high resolution of 13.8 min with high symmetry. The relative
standard deviation (RSD) for all of the system suitability parameters was less than 5%. The retention time of
Cr(VI) and Cr(III) was found to be 9.5 min and 13.0
min, respectively (Figure 6).
Validation Studies
Following optimization of the HPLC conditions, the
method was validated in accordance with ICH guidelines.
2811
VALIDATION PARAMETERS
Figure 4. Mass spectra of a) Cr-APD complex and b) Cr-POS complex to calculate molecular weight of both complexes.
Table 1. Results of chromium complex.
Sample
1
Molecular weight
Percentage yield
Percentage purity
λ max (nm)
Cr-APD complex
Cr-POS complex
Theoretical: 493
Found: 493.8
82
75
254
Theoretical: 508
Found: 506.8
85
87
256
1
Mass spectral analysis with electrospray ionization mass
spectrometry (ESI-MS).
Figure 5. Effect of pH on Cr-APD complex formation.
Table 2. Optimized chromatographic conditions used in this study.
Parameter
Chromatograph
Column
Mobile phase
Flow rate
Detection
Oven temperature
Injection volume
Optimized condition
Shimadzu Prominence LC20A
Kinetex C18, 5μ , 250×4.6 mm
Acetonitrile:Water (70:30)
0.6 mL/min
254 nm, PDA detector
50◦ C
20 μ L
Linearity and range. The linearity of measurements
was evaluated by analyzing different concentrations
of the standard solutions of Cr(III) and Cr(VI) samples. The Beer–Lambert concentration was found to
be 0.125–4 μg/mL for Cr(III) and 0.1–3.5 μg/mL for
Cr(VI). A calibration curve was constructed by plotting the average peak area against concentrations, and
a regression equation was computed. The r2 value of
Cr(III) and Cr(VI) was 0.9926 and 0.9983, respectively,
as shown in Figure 7. This method demonstrated very
good linearity for both ions at trace levels. The correlation coefficient values were found to be within the
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UMESH ET AL.
Table 3. System suitability test parameters.
Cr(III)-APD complex
System suitability
parameter
Retention time (tR )
Area
Height (mAU)
Theoretical plate
number (N)
Tailing factor (t)
∗
Cr(VI)-POS complex
Mean
% RSD
Mean∗
% RSD
13.16
861,214
90,317
26,077
0.16
4.57
4.59
0.96
9.5
1,534,667
212,529
23,808
0.12
4.74
4.42
3.33
1.07
0.22
1.01
1.38
Mean of five replicate injections (n = 5).
guideline limits for both complexes. The results
showed that an excellent correlation exists between
the peak area and concentration of ions within the
concentration range indicated in the graph shown in
Figure 7.
Precision. An intraday precision study of the CrAPD complex of both ions was carried out by estimating the correspondence responses 9 times on the same
day with 0.5 μg/mL concentration of the chromium
complex. An interday precision study was conducted by
analyzing the correspondence responses 5 times on the
next 2 d at the same concentration. The repeatability
results were found to be 3.06 and 1.82% for Cr(III) and
Cr(VI) for intraday precision and 3.29 and 3.14% for interday precision, respectively. The results obtained for
interday precision (% RSD) and intraday precision (%
RSD) for both Cr(III) and Cr(VI) were found to be less
than 5% (Table 4).
Specificity. The specificity of the analysis was studied by the resolution of the two components that eluted
closest to each other. Also, the peak purity test analysis using a PDA detector confirmed that the chromatographic peak for both chromium complexes could not
be attributed to more than one component (Figure 6).
Figure 6. System suitability parameters of chromium complexes.
Figure 7. Linearity regression plot for a) Cr(III)-APD complex and b) Cr(VI)-POS complex.
2813
VALIDATION PARAMETERS
Table 4. Precision studies of Cr (III) & Cr (VI).
% RSD
Cr ion
Concentration Cr
complex (μ g/mL)
Cr(III)
Cr(VI)
0.5
0.5
Day 1∗ Day 2† Day 3†
3.061
1.817
4.50
3.68
Average %
RSD
2.31
3.92
3.29
3.14
Total number of replicates: (n = 9)∗ and (n = 6)† .
A three-dimensional chromatographic view of both
complexes gives a single chromophoric signal, which
confirms that there was no co-elution of another impurity peak with the analyte. Commonly used excipients (glycol, propionic acid) were spiked, and noninterference was confirmed in the chromatogram. Also,
forced degradation of chromium propionate by acid and
alkali hydrolysis was completed using 50% hydrochloric
acid and 1N sodium hydroxide solution. There were no
significant changes in the elution pattern, and no new
peak was observed in the chromatogram. This confirms
that this reagent is highly specific to Cr ions. The interference of other minerals, such as Zn, Cu, Cd, and
Fe, was studied, and it was found that no complexation
reaction with APD occurred.
Accuracy. Accuracy studies were completed by
spiking known the concentration of the Cr standard solution with the chromium propionate (CrP) solution.
Percentage recovery was calculated based on the total chromium content. The recovery studies were carried out in triplicate over the specified concentration
range, and the amount of Cr(III)-APD was estimated
by measuring the peak area ratios by fitting these values to the straight line equation of the calibration curve.
Three different concentrations (80–120%) of the authentic standards were added to the 1 μg/mL solution
of chromium propionate. The resulting sample solutions
were injected, and chromatograms were recorded. The
percentage recovery range was 94–106%, well within the
±15% specification limit. The highest percentage recovery of Cr(III)-APD was found to be 100.6%, indicating
that the proposed method is highly accurate. The mean
percentage recovery obtained for the total chromium is
tabulated in Table 5.
For the Cr-POS complex, the percentage recovery
was cross verified by another analytical method using
ion chromatography; the results are shown in Table 6.
The measurement by ion chromatography is due to the
low concentration of Cr(VI) in chromium propionate,
which is well below the quantification limit. From the
foregoing determination, the percentage recovery and
the standard deviation of percentage recovery were calculated. The RSD value for both complexes was less
than 5%, which revealed that the developed method is
precise.
Limit of detection/limit of quantification. The
LOD and LOQ for Cr(III) were found to be 0.15 μg/mL
and 0.35 μg/mL, respectively, which indicates the sensitivity of the method. Similarly, for Cr(VI), LOD and
LOQ were found to be 0.07 μg/mL and 0.25 μg/mL,
respectively. The LOD value clearly suggests that the
developed method can be applied to quantify chromium
ions at trace levels in all the samples. The results were
within the specification limit. The data are tabulated
in Table 7.
Robustness. Robustness was analyzed by varying
experimental parameters such as incubation temperature and time with respect to complex formation.
No significant variation was observed at 10% deviation from the temperature (52–62◦ C) and time (15–25
min). Also, the robustness of the HPLC method was
determined by making slight changes to the chromatographic conditions, such as a change in the mobile phase
Table 5. Accuracy reading of Cr (III) by recovery studies in chromium propionate.
CrP concentration
(μ g/mL)
Concentration of
spiked Cr(III)
(μ g/mL)
Total area (1μ g/mL
of spiked CrP+
concentration)
Total concentration
of Cr(III) found
(μ g/mL)∗
Relative percentage
difference
1
1
1
1
1
1
1
1
1
0.8
0.8
0.8
1.0
1.0
1.0
1.2
1.2
1.2
1,825,457
1,707,388
1,802,451
2,020,616
1,968,854
2,002,428
2,295,049
2,347,161
2,465,136
1.82
1.71
1.80
2.02
1.97
2.00
2.29
2.34
2.46
101.34
94.78
100.06
100.96
98.38
100.05
104.26
106.63
111.99
∗
Correlation coefficient: r2 = 0.9925; equation for regression line: Y = 1E + 06x + 1355.1 (n = 3).
Table 6. Comparison of results of Cr(VI) ion estimation in CrP product using HPLC and ion
chromatography methods.
Sample
Ion chromatography (μ g/mL)
HPLC method (μ g/mL)∗
Relative percentage difference
Cr(VI)
Cr(VI)
0.29
0.26
0.26
0.28
89.65
107.9
∗
Mean replicate of three injections (n = 3).
2814
UMESH ET AL.
Table 7. LOD/LOQ of Cr complex.
Sample
Cr(III)-APD
Cr(VI)- POS
∗
LOD (μ g/mL)
LOQ (μ g/mL)
Signal/noise
% RSD∗
0.15
0.07
0.35
0.25
11.1
8.8
4.1
4.4
Mean replicate of six injections for LOQ (n = 6).
Table 8. Validation parameters.
Parameter
Absorption maxima
Linearity range (μ g/mL)
Standard regression equation
Correlation Coeffiecient (r2 )
Accuracy (% recovery ± SD)
Precision
Intraday precision (% RSD)
Interday precision (% RSD)
Specificity (% RSD)
LOD (μ g/mL)
LOQ (μ g/mL)
Cr(III)-APD
Cr(VI)-POS
254
0.125–4
y = 4,684.8x – 14,269
0.9926
99 ± 7
254
0.1–3
y = 675,387x + 12,804
0.9983
99 ± 10
3.06
3.29
4.87
0.15
0.35
1.82
3.14
0.07
0.25
Table 9. Analysis of total Cr in feed samples/mineral mixtures.
Sample
Feed sample 1
Feed sample 2
KemTRACE Cra
Cr blendb
a
b
AAS (μ g/mL)
HPLC (μ g/mL)
Relative percentage difference
1.8
1.5
8.3
2.15
1.63
1.48
7.65
2.13
90.56
98.6
92.1
99.06
kemTRACE Cr contains 5 mineral supplements, vitamins, silica.
Mixture contains CrP-118 mg, 1g CaCO3 , and silica.
composition, flow rate, and column temperature by 5%.
A slight shift in the retention time to 1.5 min was observed, with similar reproducibility for both ions. This
method is highly robust with a slight variation in experimental conditions. No marked differences were observed in the chromatogram’s elution pattern. The complete validation results for both chromium complexes
are tabulated in Table 8.
Application to Mineral Mixtures/Feed
Samples
The HPLC method developed in this study was
used to determine the total chromium in mineral mixtures and feed samples. Mineral mixtures containing the
Cr(III) ion were digested, using 50% HCl, on a hot plate
for 10 min at 100◦ C. The sample was then filtered in
Whatman Grade 1 filter paper (Sigma-Aldrich, Bangalore, India), and recovery was done in accordance with
the general procedure. In the case of feed samples, a
known concentration of Cr(VI) was spiked for recovery analysis. The feed samples were extracted using a
1:1 methanol water system at 50◦ C and 150 rpm. The
sample was filtered and centrifuged, and analysis was
completed in a manner similar to the general procedure of chromium analysis. The percentage recovery of
chromium in feed (n = 3) and mineral mixtures was
well within the specification limit, and the results are
tabulated in Table 9. Also, in the mineral mixtures the
interference of fillers and other excipients (CaCO3 and
silica) in the Cr blend did not interfere with the analyte concentration, and percentage recovery was calculated (Table 9). The results of the feed matrix sample
and mineral mixtures analyzed show that the developed procedure is useful for determining both Cr(VI)
and Cr(III) in solid matrices. The interference of other
inorganic ions with APD was found to be very minimal
and favors the application of this method as a quality
control tool for the quantification of chromium.
CONCLUSIONS
In the present study, the complexation reaction of
Cr(III) and Cr(VI) with APD was briefly discussed,
and the HPLC method was validated for quantification.
This was the first attempt to quantify chromium ions in
a solid matrix like organic chelates, mineral mixtures,
and feed samples. The method was validated with acceptable performance of linearity, precision, repeatability, accuracy, and robustness in accordance with ICH
guidelines. More importantly, the optimized method
was successfully applied to analyze the chromium content in all the formulations.
ACKNOWLEDGMENTS
The authors thank SAIF, Indian Institute of Technology, Madras, India, for help with the NMR and EPR
VALIDATION PARAMETERS
measurements and Dr. Mitch Poss and Dr. Rick Myers
for their critical reading of the manuscript.
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