An HPLC Method for detection of Anti-inflammatory Drugs in Bone and Cartilage health supplements

 

Sevil Banay Razi1, Farzaaneh Zaaeri2, Hamid Akbari Javar2*

1Department of Drug Quality Assurance, Faculty of Pharmacy, Pharmaceutical Sciences Branch,

Islamic Azad University, Tehran, Iran.

2Department of Pharmaceutics, Faculty of Pharmacy, Tehran University of Medical Sciences, Tehran, Iran.

*Corresponding Author E-mail: akbarijo@tums.ac.ir

 

ABSTRACT:

Many bone-building and cartilage repair supplements are available in the market and, many people especially the elderly people use these supplements. Manufacturers claim various therapeutic properties for these types of supplements. Therefore, it is likely that these products are included with chemical and anti-inflammatory drugs that are not listed in labels and brochures. There is not reported using HPLC method to check for chemical and anti-inflammatory drugs in bone and cartilage supplements, by the competent authorities. In this research, a valid, simple, affordable and selective method launched to quantify and identify some corticosteroids in bone and cartilage supplements. This method involves the extraction of the case from supplements using ethanol and injection directly to high-performance liquid chromatography (HPLC). The effect of different variables such as mobile phase, column type, pH and other variables was investigated. Optimum conditions including water-acetonitrile mobile phase with a ratio of 55:45, ODS column, column temperature of 25°C, injection volume of 20 microliters, detector wavelength of 254 nm and the mobile phase flow rate 1ml/min were obtained. With this HPLC analysis method, cartilage repair products and bone-building supplements can be simply analyzed and the proportion of infected or free corticosteroids can also be identified.

 

KEYWORDS: Cartilage repair supplement, Bone-building supplement, corticosteroids, HPLC analysis, prednisolone, hydrocortisone

 

 

 

INTRODUCTION:

Many bone and cartilage repair supplements are sold in the market and, elderly people use for improvements in their bones and cartilages. Producers claim many therapeutic advantages for these products such as fast recovery of bone and cartilage. Hence, there is an uncertainty of prohibited anti-inflammatory and chemical drugs that are included in products but not mentioned in labeling.

 

Food supplements are used for delivery of some essential food ingredients that are not provided by usual food regimens. Supplements generally contain vitamins, minerals, fiber, fatty acids and aminoacids. Food and Drug Administration (FDA) defines food supplements as food, although they may be classified as drug or other products. More than 50,000 kinds of food supplements are available around the world. More than half of adults in the united states usually use food supplements especially multi vitamins[1].

 

Most of the supplements in the market that claim fortification and recovery of bones and cartilages or joints pain reduction, contain Glucosamine and Chondroitin, minerals containing calcium, magnesium, manganese, zinc, copper, phosphorus, vitamins D, C, K and fatty acids such as Omega 3. Many elderly people certified that their pain has decreased after using these supplements. Therefore, there is a high possibility of some chemical substances and anti – inflammatory drugs such as corticosteroids to be included in the composition of these supplements[1– 4].

 

Corticosteroids are a group of hormones that act against inflammation in the body and have anti-inflammatory properties. These hormones suppress the immune system to reduce inflammation and pain[2,5]. A lot of drugs based on endogenous corticosteroids with similar structure and pharmaceutical action have been built and widely used in allergy, autoimmune diseases and inflammation. Examples include betamethasone, dexamethasone, hydrocortisone, triamcinolone, methyl prednisolone, prednisone, fludrocortisone, fluticasone, and so on. These drugs are produced and consumed in the form of injections, oral drops, ointments and nasal sprays. Corticosteroid medications ​​are mostly given in the higher value than the normal amount in the body. Higher concentrations of these compounds have potent anti-inflammatory effects and alleviate symptoms of arthritis, asthma and inflammatory bowel disease[2– 7].

 

Longtime usage of these pharmaceuticals may increase blood sugar in susceptible people and leads to Diabetes[8]. Osteoporosis is a well-known side effect of long time administration of corticosteroids[9]. Depression and high blood pressure are other complications. When patients use corticosteroids, adrenal glands decrease or stop natural cortisol secretion levels. Therefore, corticosteroids have many adverse side effects such as the accession of depression, hallucinations, mania and manic attacks as well as mental disorders[2]. The effect on the body's fluid balance and electrolytes, leads to salt and water retention in the body and ultimately edema[8]. The effect on fat distribution results in the subsequent accumulation of fat in certain areas of the body such as back and neck[10]. High blood pressure, high blood glucose (hyperglycemia), diabetes and hyperlipidemia, are known as strong factors for cardiovascular disease and heart attacks[11]. Bone loss and osteoporosis, as well as rupture or tendon damage in high doses are other side effects[9,12]. Reduced and weakened layers of the gastrointestinal mucosa, especially gastric ulcers are caused and exacerbated[13]. Intraocular high pressure, glaucoma and cataract are also observed[14]. Therefore, corticosteroids are usually medicated in combinations with other safer drugs or as prodrugs in order to release drugs only near the target site, so that mentioned side effects are decreased[15– 17].

 

Products that include combinations of Glucosamine and Chondroitin make effective help in the treatment of pain in arthritis. Other applications including the treatment of rheumatoid arthritis and osteoporosis have not been proven by research[18]. In the early 1980s, in a cross-sectional study, 30 hospitalized patients with knee osteoarthritis were randomized to use placebo, a combination anti-arthritic medication or glucosamine sulfate 1500mg daily for three weeks. The group that received treatment with glucosamine sulfate showed significant improvements in pain, joint tenderness and edema, whereas in the control group, there were no such improvements[19]. The medicinal use of these products is not approved by FDA (Food and Drug Administration) and doctors are not allowed to prescribe people as a medication. Glucosamine and Chondroitinare are often sold in drug stores as an herbal supplement although there are no production standards for their preparation as plant products. Some of these supplements are containing toxic metals and chemical drugs[20].

The existence of corticosteroids and chemical agents in food supplements is contrary to regulations of the WHO (World Health Organization). Accordingly, development of a method to identify chemical drugs and the quality of cartilage maker and bone-building supplements is necessary.

 

A number of studies have been conducted to evaluate and analyze combinations of corticosteroids in different samples such as medications, hair, plasma, urine and food.UV spectrophotometry, HPTLC (High performance Thin Layer Chromatography) and HPLC (High performance Liquid Chromatography) methods have been used for analysis of corticosteroids[21,22]. Some of different methods are listed in table 1.

 

 

Table1.Example studies in the field of analysis of corticosteroids.

Reference
Sample
Steroid

Instrument/Method
Study
[23]
human urine
Nandrolone
Testosterone
GS / MS extraction by n- pentane and MSTFA* / NH4I / Ethanethiol
Identifying and measurement of anabolic steroids in dietary supplements
[24]
human plasma
Cortisol, cortisone, prednisolone, prednisone
LC–MS/MS extraction by acetonitrile: water: formic acid (32:68:0.1, v/v/v)
Quantitative analysis of cortisol, cortisone, prednisolone and prednisone in human plasma
[25]
human urine
Triamcinolone, cortisone
Prednisone, dexamethasone
methyl testosterone formoterol
LC-MS / MS extraction by 0.1% acetic acid in water and acetonitrile
Detection of corticosteroids, anabolic steroids and ß2-Agonist during Athens 2004 olympic games
[26]
human urine
prednisolone

prednisolone metabolites
LC-MS / MS extraction by water, ammonium formate, acetonitrile with formic acid (0.01%)

Detection and characterization of prednisolone metabolites in human urine

[27]
human plasma
methyl prednisolone
LC-MS / MS extraction by
10 mM ammonium formate buffer and acetonitrile (35:65, v/v)
Assay of methylprednisolone in human plasma and its pharmacokinetic application
[28]
human plasma

human urine human saliva
prednisone
dexamethasone
cortisone
cortisol
UHPLC-MS / MS extraction by ammonium acetate in water and ammonium acetate in methanol both with 0.1% formic acid.
Determination of glucocorticoids in human plasma, urine and saliva
[29]

rat plasma
prednisolone acetate
methyl prednisone acetate

 
LC–ESI MS/MS extraction by 50% water containing 0.01% formic acid and 50% acetonitrile
A rapid, sensitive and specific method for the simultaneous detection of corticosteroids
[30]
human urine
 
Beclomethasone,
betamethasone
dexamethasone
prednisolone, prednisone
methyl prednisolone
LC-MS / MS extraction by 1% Formic acid and acetonitrile
 
Detection of synthetic corticosteroids
[31]
human plasma
Prednisolone, prednisone
Cortisol, cortisone

GC–MS derivatization by heptafluoro-n-butyric anhydride (HFBA)
Simultaneous determination of corticosteroids in plasma
*MSTFA: N-methyl-N-trimethyl silyl trifluoro acetamide
**LC–ESI MS MS: Liquid chromatography–electrospray-tandem mass spectrometry

 

 

As a result of discussed subjects, specification of a method for identification of anti-inflammatory and chemical drugs for quality control of bone and cartilage fortifier supplements is needed. In this study, we aimed to practice and validate a standard and simple HPLC method for identification and determination of chemical unwanted corticosteroids in bone and cartilage regeneration supplements. We looked for a separation method based on gas or liquid chromatography followed by a spectrophotometric method for specification and identification of the slightly ingredients. We explained a standard method using the similar articles as well as international standards in this field.

 

MATERIAL AND METHODS:

Full experimental work conducted to set up an accurate, inexpensive and easy method. All the laboratory analyses done based on standard Prednisone and Hydrocortisone.

 

1. Materials and standards:

Standard prednisone and hydrocortisone were obtained from Iran hormone industries. HPLC grade methanol and acetonitrile were purchased from Merck. HPLC grade purified distilled water was used.

 

2. Instruments:

High-performance liquid chromatography (HPLC) system manufactured by DIONEX company with a gradient pump model Ultimate 3000 equipped with autosampler 100 microliter injection in maximum was used. There was a separate oven to adjust the temperature of the column. UV detector model UVD170U made by company DIONEX, column C18 (ODS= Octadecylsilane) (460 mm × 250 mm) containing particles with a diameter of 10µm Manufactured by Thames Resiek company and the software Auto Chrom 2000 for recording chromatograms were used. UV spectrophotometer model UV-265FW manufactured by Shimadzu was used for concentrations evaluation. Digital scale Model B154 Manufactured by Mettler Toledo company with a precision of 0.1mg, digital scale manufactured by Shimadzu with a precision of 0.0001mg, ultrasonic bath model 4200 Manufactured by Solte company, pH meter manufactured by Mettler Toledo company and centrifuge model D72 made by Andreas Hettich company were used. Millipore filtration system with a vacuum pump and water Purification system model Basic 360 coupled with the Max-ultra 354 Aqua Manufactured by Younglin company were used to remove particles from solvents and make HPLC grade water, respectively.

 

3. Optimization of the extraction method:

To extract corticosteroids from cartilage repair (bone-building) supplements, we used solvents that dissolve these compounds as well but not dissolve other components. In another word, excipients should be poorly soluble or insoluble in the solvents. We examined solvents such as methanol, ethanol, acetonitrile, and chloroform and investigated the extraction process. Standard hydrocortisone and prednisolone solutions in a concentration of 10μg/ml were prepared by each HPLC grade solvent and centrifuged for 10 min. Supernatants were diluted 10 times with solvents and their absorptions were determined by UV spectrophotometer. In the extraction process, choosing an efficient solvent is the most crucial part of the process because the solvent must solve goal substance as well but does not solve other compounds and excipients in the sample matrix. Since the compounds found in cartilage maker (bone-building) supplements are dissolved in water, solvents such as acetonitrile, chloroform, methanol and ethanol were used. In order to achieve the most appropriate solvent, extraction process was performed and absorption spectra were provided.

 

4. Selection of the optimum condition for HPLC process:

In this study, we aimed to achieve an optimal method of high-performance liquid chromatography (HPLC) to identify and quantify the anti-inflammatory compounds (hydrocortisone and prednisone) in cartilage maker supplements. According to previous studies to identify anti-inflammatory compounds by HPLC method, in order to view the appropriate peak for these compounds, the mobile phase of water: methanol in a gradient washing was used and the percentage of mobile phase portions was changed from 50:50 (water: methanol) to 100% methanol. Other conditions were set as follows: C18 column with a length of 12.5cm, a diameter of 4.5 mm and particle size of 10 micrometers, UV detector wavelength of 254nm, flow rate of 1ml/min for mobile phase, column temperature of 30oC and injection volume of 20ml. Standard solutions of prednisolone and hydrocortisone with a concentration of 0.5μg / ml were prepared and injected[32–34]. To achieve a fit peak with the best area in an HPLC analysis, various parameters including column, type and percentage of mobile phase, UV detector wavelength, column temperature, injection volume and flow rate of mobile phase can be changed[35– 38].

 

To select a proper column, a variety of factors including properties of substance such as solubility, chemical nature of the functional groups and molecular weight should be regarded. It must be examined whether the species are selectively separable on the stationary phase. In this study, standard solutions of hydrocortisone and prednisolone in a concentration of 50μg/ml with C8 and C18 columns from different companies were studied to identify anti-inflammatory compounds and acquire the most appropriate peak. To select a proper column, a variety of factors including properties of species such as solubility, the chemical nature of functional groups in any desired species and molecular weight should be considered. It must be examined whether the species can be separated on the selected stationary phase. Chemical structure of the corticosteroids or anabolic steroids such as hydrocortisone and prednisone contains a lipophilic base with four carbon rings and one or more polar groups resulting being insoluble in water or low soluble. As a result, column chromatography with C18 stationary phase and normal phase chromatography are recommended to identify and isolate them. Columns have large differences in retention of materials due to the different construction of active silanol groups.

 

Mixtures of water and acetonitrile are the most common mobile phases for liquid chromatography with high efficiency. Acetonitrile and methanol have different chromatographic characteristics. Acetonitrile has low viscosity, low UV absorption, good kinetics which makes sharper peaks, more efficient washing that leads to lower consumption of solvent, and less back pressure for acetonitrile-water mixtures in comparison with mixtures of methanol-water. Methanol is odorless and less toxic than acetonitrile which causes better and safer working conditions. Salts have better solubility in it and the risk of deposit formation is lower. It is suitable for isolation of bases in alkaline pH condition. On the other hand, the polarity of the mobile phase is effective in separation of species and chromatographic washing. In reverse phase systems with a non-polar C18 stationary phase, non-polar species have longer retention times and their exit time from the column is altered by changing the ratio of mobile phase. Among three solvents of water, acetonitrile and methanol, dipole moment in water is stronger and as a result water has the highest and methanol has the lowest polarity. But water is not a good washing solvent for corticosteroids causing delayed elution, so the percentage of organic solvent should be increased.

 

Changes in column temperature cause changes in the chromatographic parameters (viscosity, kinetics, efficiency) and chemical or thermodynamic changes (enthalpy of adsorption, retention time and selectivity). An increase in temperature from 30°C to 40°C causes peaks move at different speeds toward the beginning of the chromatogram and the retention time and selectivity is slightly reduced but the column pressure is increased. In this study, standard solutions of hydrocortisone in concentration of 100μg/ml and prednisolone in concentration of 20μg/ml were prepared and different temperatures from 25 to 40°C were applied on the column. The peaks obtained and the effect of temperature on the area under the peak and retention time of samples was analyzed.

 

Ideal flow rate for this study was considered 1 ml/min. To confirm method robustness and the optimal flow rate, flow rates from 0.9 to 1.1ml/min using standard solutions of hydrocortisone 100μg/ml and prednisone 20μg/ml were applied. Each standard solution was individually injected three times to the HPLC system and the effect of flow rate on the retention time and the area under the peak of standard solutions and control samples was studied. According to the conducted review on chromatograms, applying the flow rate of 1/1 ml/ min for both standard and control samples reduces retention time of the samples; the sample and the solvent peaks may interfere, and the area under the peak may show a decline. While applying a flow rate of 0.9ml/min for both solutions increases the retention time of the sample there by the sample is removed later and the area under the peak is also enlarged.

 

To confirm an appropriate wavelength, wavelengths of 250nm and 260nm using standard solutions were applied and retention time and area under the peak of each method were studied.

 

In order to perform quantitative analysis and measurement of anti-inflammatory compounds in these supplements, calibration curves for each compound was separately plotted. To plot calibration curve of hydrocortisone, solutions with different concentrations from 10 to 130μg/ml were prepared and the best concentration range for plotting the curve was obtained. The appropriate graph with concentrations in the range of 70 to 130μg/ml was plotted. 5 solutions at concentrations of 70, 80, 130, 115, 110μg/ml were injected into the HPLC system. Each sample was analyzed three times and a calibration curve was plotted. To plot calibration curve of prednisolone, solutions with different concentrations from 1 to 13μg/ml were prepared and the best concentration range was obtained. The appropriate graph with concentrations in the range of 7 to 13μg/ml was plotted. 5 solutions at concentrations of 7, 9, 10, 12, 13μg/ml were prepared and injected into the HPLC system. Each sample was analyzed three times and a calibration curve was plotted.

 

5. Method Validation:

After the best analysis method was achieved, it should be validated by the conditions and standard materials. Validation methods were applied to investigate validation parameters for evaluation of hydrocortisone and prednisolone in bone-building and cartilage repair supplements.Validation factors including selectivity, linearity, precision, accuracy and limit of detection were measured[32–40].

 

Linearity within the range of analysis should be confirmed. Linearity is determined by visual inspection of calibration curve plotted based on analytical signals against samples concentrations. After the initial ensuring linearity of the graph, a closer look should be done by an appropriate statistical method. To evaluate the linearity of hydrocortisone analysis method, standard solutions with concentrations of 70, 85, 90, 100, 110, 115 and 130μg/ml were prepared and each solution was injected twice into the HPLC system and linearity curve was plotted. For prednisolone the method was conducted in the same way, but with different concentrations of 7, 8, 9, 10, 11, 12 and 13μg/ml[35].

 

Precision of an analysis method indicates the degree of coordination among the results of separate tests in condition that multiple samples are taken from a homogeneous sample. Precision of an analysis method is usually explained by standard deviation (SD) factor and relative standard deviation (RSD) that is also stated as coefficient of variation (CV). Precision may be illustrated as reproducible precision or reproducibility of the method of measurement under normal conditions of analysis. Precision of the analysis method is determined at two levels. Repeatability was specified by determination of technique precision in one day, in one laboratory and by one examiner. This way is also called Intra-day evaluation of precision. Reproducibility was elucidated by measurement of precision in different days, in different laboratories by different devices and testers which is also called the Inter-day precision. Precision of the method was carefully specified for hydrocortisone and prednisolone analysis by HPLC.

 

The real amount of active ingredient in a pharmaceutical formulated product is nominated as accuracy. Accuracy is verified by adding a known amount of substance to the excipient materials in the range of analysis method which can be precisely determined. If it is not possible to provide samples of the excipients, another acceptable way to determine accuracy is that known amounts of substance is added to pharmaceutical product and the results of analysis of substance is compared with the results of another proposed method that its accuracy has been proven. Hence for the determination of accuracy of the method, standard solutions of hydrocortisone with concentrations of 90 and 110μg/ml and prednisolone with concentrations of 8 and 11μg/ml were added to the samples and injected 3 times into the HPLC system. The recovery for each concentration was calculated and reported as accuracy.

 

According to ICH guidelines, signals measured with low concentrations of certain species should be compared with blank samples (control). The lowest concentration of the sample which is detected and differentiated from background noise (the blank), is introduced as detection limit. Signal to noise ratio of 3: 1 or 2: 1 is accepted. Detection limit is not involved in quantification of samples. (LOD =Signal /noise)

 

To determine the quantification limit of method, according to ICH guidelines, signals for low concentrations of samples should be compared with blank samples (control). The lowest concentration which is reliably determined, is introduced as limit of quantification. Signal to noise ratio of 10: 1 is considered as LOQ that enables reasonably recognition of the concentrations. (LOQ = Signal /noise)

 

Proof of selectivity or specificity requires in determining the amount of impurities with appropriate accuracy and precision along showing that the process of analysis is not affected by the presence of impurities. In practice, this can be achieved by adding raw pharmaceutical material or product to a mixture of appropriate amounts of impurities and excipients showing that the results of the quantification are not influenced by the presence of foreign substances. Selectivity of the method for determination of hydrocortisone and prednisolone was evaluated by adding 1ml of 5μg/ml standard solution of hydrocortisone to 100mg of product powder which is free of any anti-inflammatory drug. Methanol was added to bring the volume and the mixture was stirred to be completely uniform. Then, it was centrifuged for an hour and the supernatant was tested. This method was similarly done for prednisolone[35,36].

 

Three types of powdered cartilage supplements in the market were tested. 1 g of each sample was extracted about drug by solvents. The resulting solution was injected directly into the HPLC system.

 

RESULTS AND DISCUSSION:

Differences in extractions by the four solvents represented that both chloroform and acetonitrile do not show acceptable separated spectra for substances. Ethanol and methanol both are suitable solvents to extract both substances but ethanol as a clean, cheap and safe solvent is preferred (fig.1).

 

Figure1. The absorption spectra of prednisolone and hydrocortisone extracted by different solvents: prednisolone extracted by acetonitrile(A), chloroform(B), methanol(C), ethanol(D) and hydrocortisone extracted by acetonitrile(E), chloroform(F), methanol(G), ethanol (H).

 

The column (C18, 250mm, 4.5mm and 10μm) from Thames Restek Ltd UK was used to find optimum conditions. According to previous studies, water-methanol mobile phase in gradient mode was used at first. Prednisolone and hydrocortisone peaks were appeared separately in this condition. Since the peaks had low height, the reproducibility of results was not observed in subsequent injections declaring that this type of mobile phase is not appropriate to identify prednisolone and hydrocortisone by this method[36,37]. At this stage, due to the chemical nature of species and instrumental conditions, the mobile phase mixture was changed to water: acetonitrile. Therefore, different percentages of the two solvents were studied and ratios of 45:55 and 55:45 (reducing the amount of acetonitrile and increasing the percentage of water) were analyzed. It was observed that by increasing the percentage of aqueous solvent, the retention time for hydrocortisone and prednisolone increases; while by reducing the percentage of organic solvent, the retention time, the area under the peak and the resolution of peaks are optimal. Thus 45% of acetonitrile according to tests was carried out. Chromatograms related to the ratios of mobile phase for prednisolone and hydrocortisone standards are showed in figure 2. The best ratio of the two solvents, according to the parameters of retention time and peak area was selected 55:45 (water: acetonitrile).

 

Figure2. Standard chromatogram of prednisolone and hydrocortisone in different ratios of solvents: Standard chromatogram of prednisolone in mobile phase with the ratio of water: acetonitrile 65: 35(A), Standard chromatogram of hydrocortisone in mobile phase with the ratio of water: acetonitrile 65: 35(B), standard chromatogram of prednisolone in mobile phase with the ratio of water: acetonitrile 55: 45(C), standard chromatogram of hydrocortisone in mobile phase with the ratio of water: acetonitrile 55: 45(D).

 

As regards a decrease in retention time depends on the sample and the mobile phase, the results for effect of rising the temperature on retention time of hydrocortisone and prednisolone standards showed that the best column temperature for analyzing the samples is 30 °C. The effect of rising the column temperature was also tested on the area under the peak and it was found that the column temperature has no effect. Chromatograms of standard concentrations of drugs in 30°C column temperature are shown in figure 3.

 

Figure3. Chromatograms of prednisone and hydrocortisone standards in different column temperatures: Chromatograms of prednisolone standard in column temperature of 30°C (A), Chromatograms of hydrocortisone standard in column temperature of 30°C (B), Chromatograms of prednisolone standard in column temperature of 40°C (C), Chromatograms of hydrocortisone standard in column temperature of 40°C (D).

 

As a result of the study of retention time and acceptable peak shape, optimum flow rate was determined 1ml/min. Average area under the peak and retention times for drugs are depicted in table 2.

 

Table2.Evaluation of retention time and area under the peak for drugs in different flow rates

Area under the peak

Retention time (min)

 

0.9

1

1.1

0.9

1

1.1

Flow rate (ml/min)

27.32

25.26

10.88

4.37

3.28

3.55

Prednisolone

75.06

69.52

61.33

9.65

6.52

7.55

Hydro

-cortisone

 

Maximum absorption for prednisolone and hydrocortisone was observed at about 250 nm. Using UV detector in wavelengths of 250 and 260 nm for detection of hydrocortisone and prednisone standards showed similar reduction in the area under the peak than 254 nm. According to the results (table 3), a wavelength of 254 nm is desirable because the retention time and area under the peak are desirable and there is no interference between sample and solvent.

 

Table3. Evaluation of retention time and area under the peak for drugs in different Wavelengths

Area under the peak

Retention time (min)

 

260

254

250

260

254

250

Wavelength (nm)

342.5

518.7

285.3

4.3

3.9

4.2

Prednisolone

581.1

654.6

521.1

8.9

8.4

9.1

Hydro

-cortisone

 

Standard calibration curve of prednisolone was plotted using standard solutions in the range of 7 to 13μg /ml. Standard calibration curve of hydrocortisone was plotted using standard solutions in the range of 70 to 130μg/ml. The manner and linearity of the calibration curves with the deviation of points of R> 0.99 represents a linear correlation for the method in used concentrations (fig.4).

 

Figure4. Calibration curve and linearity evaluation for prednisolone (A) and hydrocortisone (B) assay

 

Standard solutions of hydrocortisone and prednisolone were injected into HPLC system to study precision of within day (intra-day) and between days (inter-day) working. The results were arranged in table4.

 

Table 4. Results of within day and between day precision study for prednisolone and hydrocortisone: intra-day precision study for prednisolone (A), hydrocortisone (B), inter-day precision study for prednisolone (C), hydrocortisone (D)

Concentration

(µg/ml)

AUC

Sample 1

AUC

Sample 2

AUC

Sample 3

 

RSD%

Sample 1

Sample 2

Sample 3

20

25.290

24.381

24.535

First

day

0.074

0.069

0.038

20

25.288

24.319

24.695

Second day

0.092

0.058

0.032

20

25.257

24.407

24.696

Third day

0.083

0.063

0.023

Mean

25.278

24.369

24.642

Mean RSD%

0.083

0.063

0.031

SD

0.019

0.168

0.093

RSD%

0.074

0.689

0.377

A

C

 

B

D

 

Concentration

(µg/ml)

AUC

Sample 1

AUC

Sample 2

AUC

Sample 3

 

RSD%

Sample 1

Sample 2

Sample 3

100

69.721

72.282

69.463

First day

0.023

0.090

0.058

100

69.393

72.423

69.545

Second day

0.031

0.095

0.060

100

69.140

72.374

69.502

Third day

0.027

0.011

0.062

Mean

69.711

72.354

69.503

Mean RSD%

0.027

0.069

0.060

SD

0.016

0.066

0.041

RSD%

0.023

0.090

0.058

 

 

Standard solutions of two drugs with different concentrations in the linear range were injected into the HPLC system. Accuracy was determined by the recovery percentage of species. The obtained results were located in the range of 97% -103% (table 5).

 

Table 5. Results of accuracy verification for prednisolone (A) and hydrocortisone (B) standards

Concentration

(µg/ml)

A

Recovery percent

(% R)

Peak area

Mean

8

38.630

38.599

100.07

8

38.658

8

38.511

11

55.259

55.353

101.05

11

55.488

11

55.277

 

Concentration

(µg/ml)

B

Recovery percent

(% R)

Peak area

Mean

90

39.292

39.158

102.09

90

39.129

90

39.054

110

47.451

47.635

100.01

110

47.569

110

47.885

 

The lowest concentrations of standard solutions of prednisolone and hydrocortisone were identified and detected equal to 0.001μg/ml and 0.02μg/ml, respectively. Signal to noise ratios for prednisolone and hydrocortisone in mentioned concentrations were calculated 5.55 and 3.09, respectively, that are in the acceptable range of ≥ 3 and ≤ 10. The lowest concentrations of prednisolone and hydrocortisone standards were measured equal to 0.01μg/ml and 0.2μg/ml, respectively. Signal to noise ratios for prednisolone and hydrocortisone in mentioned concentrations were calculated 12 and 16, respectively, that are in the acceptable range of ≥ 10.

 

Solutions resulted from the extraction processes were injected into the HPLC system, and at wavelength of 254nm two peaks related to prednisolone and hydrocortisone were appeared. Chromatograms obtained from analyzing standard solutions of hydrocortisone and prednisolone, cartilage supplements without hydrocortisone and prednisolone (placebo) and cartilage supplements containing hydrocortisone and prednisolone (Spiked) at the wavelength of 254nm showed that the two drugs can be separated by a resolution coefficient of >3 and do not interfere with other ingredients (fig.5).

 

Figure 5. Results for analyzing supplements at 254 nm to study specificity of the method: standard solution of prednisolone (A), cartilage supplement without prednisolone (B), cartilage supplement containing prednisolone (C), standard solution of hydrocortisone (D), cartilage supplement without hydrocortisone (E), cartilage supplement containing hydrocortisone (F).

 

 

Analysis of real samples of cartilage supplements (bone-building supplements):

Three cartilage repair supplements were analyzed by described HPLC procedure at the wavelength of 254nm to examine whether the claims on the labels of these supplements that are free of any chemical compounds, are true or not. Chromatograms derived from the supplements at 254nm are shown in Figure 6.

 

Figure 6. Chromatograms obtained from the analysis of real samples of cartilage repair supplements in the market (Glucosamine capsules) for prednisolone and hydrocortisone

 

According to the chromatograms, to determine the amount of active ingredients of prednisolone and hydrocortisone in three glucosamine capsule samples, the average area under the curve of prednisolone and hydrocortisone in real sample solutions, were determined and the amount of the compounds found in each sample was calculated using calibration line equation and reported in µg/g value (table 6).

 

Table 6. The results for assay of prednisolone and hydrocortisone in three samples of glucosamine capsule in the market

Corticosteroid

Sample A (µg/g)

Sample B (µg/g)

Sample C (µg/g)

Prednisolone

0.27

0

0.25

Hydrocortisone

2.37

0.59

1.04

This study aimed to achieve an optimal HPLC method to identify and quantify the anti-inflammatory compounds (prednisolone and hydrocortisone) in cartilage repair products. According to research done, parameters such as column type, the wavelength of detector, the percentage of each solvent in mobile phase, column temperature, flow rate and injection volume for the analysis of prednisolone and hydrocortisone were optimized. Optimum conditions of parameters are presented in the table 7.

 

Table 7. Optimum values ​​for analysis of prednisone and hydrocortisone by HPLC method

Optimized condition

Parameters influencing in the analysis

C18: 25cm/4.5 mm/10µm

 

Column: length/diameter/stationary phase particles diameter

254 nm

Wave length of detector

water: acetonitrile 55: 45

Mobile phase

30oC

Column temperature

1 ml/min

Flow rate

20 µl

Injection volume

 

CONCLUSION:

In this study an HPLC analysis method for determination of the amount of corticosteroids (prednisolone and hydrocortisone) in cartilage repair supplements was tested. The method is simple, rapid, selective, affordable and valid. The method of extraction and sample preparation are simple and fast as well as the method is highly efficient. The method showed acceptable accuracy and precision in an appropriate linearity range therewith low detection and quantification limits, for the analysis of corticosteroids in cartilage repair supplements. In this method, the matrix of supplements (excipients) does not interfere to the evaluation of corticosteroids. The developed method was accepted as a valid applicable instruction for evaluation of corticosteroids in cartilage repair products and validated as a Standard Operating Procedure (SOP) by Iran Reference Food and Drug Control Laboratories. As a result, evaluation of cartilage repair (bone-building) supplements that are imported to the country and estimation of products infected with corticosteroids is accomplished by this method. This work is the first study done toward the assessment of dietary cartilage repair (bone-building) supplements regarding to the determination of corticosteroids.

 

ACKNOWLEDGEMENT:

This work was supported by Iran Food and Drug Control Laboratories by the equipment and materials but it did not receive any grant from funding agencies in the public, commercial, or not-for-profit sectors.

 

CONFLICT OF INTEREST:

The authors announce that there is no conflict of interest about article.

 

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Received on 12.10.2019       Modified on19.11.2019

Accepted on 28.12.2019      ©Asian Pharma Press All Right Reserved

Asian J. Pharm. Ana. 2020; 10(2):67-76.

DOI: 10.5958/2231-5675.2020.00012.5