Quality Control of Non-Sterile Drug Product According to United States’ Pharmacopeia Instruction

Gholizadeh-Hashjin, Aiesheh; lotfipour, farzaneh; Hallaj-Nezhadi, Somayeh

doi:10.30699/ijmm.13.5.321

year 13, Issue 5 (November - December 2019) Iran J Med Microbiol 2019, 13(5): 321-345 | Back to browse issues page

‎ 10.30699/ijmm.13.5.321

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Gholizadeh-Hashjin A, lotfipour F, Hallaj-Nezhadi S. Quality Control of Non-Sterile Drug Product According to United States’ Pharmacopeia Instruction. Iran J Med Microbiol 2019; 13 (5) :321-345
URL: http://ijmm.ir/article-1-966-en.html

Quality Control of Non-Sterile Drug Product According to United States’ Pharmacopeia Instruction

Aiesheh Gholizadeh-Hashjin¹

, Farzaneh Lotfipour¹

, Somayeh Hallaj-Nezhadi²

1- Food & Drug Control Department, Pharmacy Faculty, Tabriz University of Medical Sciences, Tabriz, Iran
2- Food & Drug Control Department, Pharmacy Faculty, Tabriz University of Medical Sciences, Tabriz, Iran , nezhadis@yahoo.com

Keywords: Quality control, Non-sterile drug products, United States’ Pharmacopoeia

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Introduction

Pharmaceutical products are classified into two groups according to the microbiological point of view: 1) sterile products and 2) non-sterile products. The sterilized term refers to the products that are free of any microorganisms, their production were done under aseptic conditions, but the production of non-sterile products were not under aseptic conditions (1); they are not free from microorganisms; for this type of products legal authorities defined microbial limit ranges.
The requirements for non-sterile products acceptance depending on the legal authorities of different countries or even different pharmaceutical companies may vary slightly. "Microbial limit Test" is a name used to evaluate the microbial content of non-sterile products and has four steps: sample preparation, preliminary testing, counting and identification, each step is described in the following.
A review study from 2004-2011 revealed that 75% of the non-sterile products recalls were from OTC (over the counter) and self-care products. Most of recalls are because of (2):
• The presence of objectionable microorganisms (72%)
• Over load of microorganisms (15%)
• Errors in sterilizing equipment or diagnostic kit (7%)
• Failure of microbiological tests (5%)
• Manufacturing defects (1%)

Objectionable microorganism refers to a pathogenic opportunistic microorganism, with specific characteristics such as production of endotoxin, exotoxin, spore and so on. These microorganisms can grow under certain temperature and nutritional conditions and could affect the quality and safety of the product. Contamination at any stage of the process can represent a serious risk to the final product and must be controlled so to maintain the quality and safety of the product (3). Very few products, such as syrup and elixir, have a preservative nature. Other products contain preservatives to keep the safety of the product (4).
Failure to comply the aseptic conditions with regard to sterile products, especially in the hospital, has led to the emergence of nosocomial infections such as Staphylococcus aureus and Enterobacter in propofol - Pseudomonas aeroginosa and Enterobacter in dextrose vial (for multiple-dose purposes) which was unfortunately led to two deaths in each of these cases (5,6). A review study in 2019 showed that extemporaneous medicine which was made in hospital and community pharmacies in some cases has led to the emergence of infection such as meningitis in people consuming these products due to microbial contamination (7). A study by Hosseyni et al. in 2014 on the gastric juice production process of a pharmaceutical company in Iran indicated the presence of some unauthorized microorganisms during the manufacturing process (8) as well as a study by Mohammadi et al. warned of the presence of unauthorized microbial agents in cosmetics (9). In 2015, a study in Saudi Arabia by Ratajczak et al. investigated 1285 non-sterile products and found that in 1.87% of the non-sterile products microbiological criteria were not approved, also naturally products were more contaminated (5.7%) (10). Table 1 lists the studies that have so far tested microbial limit in non-sterile products in Iran. Due to the few numbers of such studies in Iran and also the results which has indicated that most products have not met the requirements of the Pharmacopoeia, there is a necessity for more attention to such studies domestically. Most contaminants of non-sterile raw materials and products include bacteria, mold and fungi (2). This indicates that quality control of non-sterile pharmaceutical products requires high precision at all stages of the process that include: raw materials, during manufacturing, final product and after market entry. There are some differences between the FDA (US Food and Drug Administration) guidelines and the US Pharmacopoeia (USP) (11). In this paper, microbial quality control methods for non-sterile products have been reviewed based on the latest USP revision, chapters <61>, <62> and <1111> (12) which can be used as a reliable source by researchers and practitioners in the pharmaceutical industry and laboratories.
Tests related to these chapters of the USP address how to quantify and identify mesophilic bacteria and fungi that may grow under aerobic conditions. The purpose of these tests is to investigate the properties of the drug's raw material and product in terms of microbial quality.
It is worth noting that the methods described in this section are not applicable to compounds that contain live microorganisms for therapeutic purpose. Also, the guidelines introduced in the USP deal only with target microorganisms and do not cover all the objectionable microorganisms represented by the FDA (13). Appendices 16 in British Pharmacopoeia (BP) (14), Chapters 2.6.12, 2.6.13 and 5.4.1 in European Pharmacopoeia (EP) (15) and Chapters 4.05 and 5.02 in Japanese Pharmacopoeia (16) have described microbial limit test. Recent editions of these books has been more harmonized so the differences between these books are very minor in new editions (17).

Table 1. Studies on microbial limit test in non-sterile drug products in Iran – (Institute of Standards and Industrial Research of Iran: ISIIR)

Title	Samples	Observations	Results	Experiments guidance	Year	Reference
Survey of bacteriological contamination of cosmetic creams in Iran	8 cosmetic creams both used and unused, were tested	High percentage of samples contaminated by Gram-positive bacilli, S.aureus and Gram-negative bacteria (except E. coli)	the quality of the samples did not generally meet the standards for microbial limits	FDA, USP & BP	2000	)18)
Microbial quality of some herbal solid dosage forms	20 samples include tablets, powders and capsules	TAMC>1100 cfu/mL in all samples Contamination of all samples with Salmonella Absence of other microorganism	the quality of the samples did not generally meet the standards for microbial limits	USP	2010	)19)
Microbial quality survey of sunscreen products in Iranian market	90 random samples of indoor, imported and prepared sunscreen at the pharmacy; at time 0, 3 and 6 months after opening	Contamination of Iranian, imported and pharmacy prepared samples with at least one of the unauthorized microorganisms at all times of sampling	the quality of the samples did not generally meet the standards for microbial limits	USP, ISIIR	2013	(20)
Characterization of Iranian bentonites to be used as pharmaceutical materials	10 samples of bentonite extracted from different regions of Iran	Absence of pathogenic bacteria	the quality of the samples generally meets the standards for microbial limits	USP, EP	2015	(21)
(1)	7 sunscreen creams (5 foreign creams from informal market and 2 creams as control from Iranian products)	Total microbial count is more than authorized level Contamination with Enterobacter in all samples and S. aureus in one of 7 samples	the quality of the samples did not generally meet the standards for microbial limits	USP	2015	(22)
Microbial Content in some Foundation Creams in Iran’s Market	7 creams (6 creams from informal market and 1 cream as control from Iranian market)	83.3% of samples and control sample were contaminated. Unauthorized fungal contam-ination was observed in 42.8% of samples. All samples were contaminated with pathogenic microorganisms (especially S. aureus).	the quality of the samples did not generally meet the standards for microbial limits	USP	2015	(23)

Chapter <1111>: Microbiological Examination of Nonsterile Products: Acceptance Criteria for Pharmaceutical Preparations and Substances for Pharmaceutical Use
Acceptance criteria for non-sterile pharma-ceutical products based on total aerobic microbial count (TAMC) and total yeast and mold count (TYMC) and tests for the absence of specified organisms (due to the route of administration) is gathered in Table 2.
According to Table 2, the interpretation of the results will be as follows:
If the number of 10¹cfu, 10²cfu, 10³cfu is stated in the table, the highest acceptable level of microorganisms in the counting method is 20, 200, 2000 and so on.
This table is not necessarily comprehensive and in specific cases may include the presence of other microorganisms depending on the nature of the raw material and the production process.

Table 2. Acceptance Criteria for Microbiological Quality of Nonsterile Dosage Forms and Substances for Pharmaceutical Use

Route of Administration	TAMC (cfu (colony forming unit)/mL or cfu/g)	TYMC (cfu/mL or cfu/g)	Specified Microorganism(s)
Nonaqueous preparations for oral use	10³	10²	Absence of E. coli (1 g or 1 mL)
Aqueous preparations for oral use	10²	10¹	Absence of E. coli (1 g or 1 mL)
Rectal use	10³	10²	………….
Oromucosal use	10²	10¹	Absence of S.aureus (1 g or 1 mL)
			Absence of P.aeruginosa (1 g or1 mL)
Gingival use	10²	10¹	Absence of S. aureus (1 g or 1 mL)
			Absence of P. aeruginosa (1 g or 1 mL)
Cutaneous use	10²	10¹	Absence of S. aureus (1 g or 1 mL)
			Absence of P. aeruginosa (1 g or 1 mL)
Nasal use	10²	10¹	Absence of S. aureus (1 g or 1 mL)
			Absence of P. aeruginosa (1 g or 1 mL)
Auricular use	10²	10¹	Absence of S. aureus (1 g or 1 mL)
			Absence of P. aeruginosa (1 g or 1 mL)
Vaginal use	10²	10¹	Absence of P. aeruginosa (1 g or 1 mL)
			Absence of S. aureus (1 g or 1 mL)
			Absence of Candida albicans (1 g or 1 mL)
Transdermal patches (limits for one patch including adhesive layer and backing)	10²	10¹	Absence of S. aureus (1 patch)
			Absence of P. aeruginosa (1 patch)
Inhalation use (special requirements apply to liquid preparations for nebulization)	10²	10¹	Absence of S. aureus (1 g or 1 mL)
			Absence of P. aeruginosa (1 g or 1 mL)
			Absence of bile-tolerant Gram-negative bacteria (1 g or 1 mL)
Substances for pharmaceutical use	10³	10²	………….

Chapter <61>: Microbiological Examination of Nonsterile Products: Microbial Enumeration Tests
General considerations
All tests should be carried out under sterile conditions to prevent the entry of other microorganisms during testing. If the medicinal product contains antimicrobial activity, the effect of the antimicrobial agent should be eliminated or neutralized (Table 3), if the neutralizing agents are used for this purpose their effect on non-toxicity to the microorganisms must be ensured (to avoid false negative results). If surfactants are used in the preparation of the sample, its non-toxicity to the microorganisms and its compatibility with the neutralizing agent should be determined

Table 3. Common neutralizing agents/methods for interfering substances

Potential Neutralizing Agents/Method	Interfering Substance
Sodium hydrogen sulfite (Sodium bisulfite)	Glutaraldehyde, mercurials
Dilution	Phenolics, alcohol, aldehydes, sorbate
Glycine	Aldehydes
Lecithin	Quaternary ammonium compounds (QACs), parahydroxybenzoates (parabens), bisbiguanides
Polysorbate	QACs, iodine, parabens
Thioglycollate	Mercurials
Thiosulfate	Mercurials, halogens, aldehydes
Mg or Ca ions	EDTA

First Step: Sample Preparation
The sample preparation depends on the physical properties of the drug product. For each sample, 10 g or 10 cc of the product is tested (unless otherwise specified in the relevant monograph). For liquid and solid aerosols and transdermal patches 10 aerosol containers or 10 patches should be tested. The dilution of the samples should be such that the possible errors are minimized and the errors are predictable, so the method should be validated (24).
some specific cases the amount to be tested may be reduced.

Second step: Preliminary Test, Bacterial Inoculation and Dilution
The purpose of preliminary test is to find out if the preservation of samples has been removed in first step or not. Thus, sufficient amounts of bacterial suspension were added to the sample and control (without sample material) to produce less than 100 cfu bacterium.
If the antimicrobial effect of preservative persists, re-addition of bacterial suspensions should be performed after neutralization, dilution, or repeated filtration.

Filtration
The size of membrane filters should be less than 0.45 µm. For the determination of TAMC, the filter membrane is transferred to the surface of Soybean–Casein Digest Agar and for the determination of TYMC the membrane is transferred to the Sabouraud Dextrose Agar surface. The plate is incubated according to the conditions in Table 4 and then counted.

Table 4. Preparation and Use of Test Microorganisms

Suitability of Counting Method in the Presence of Product		Growth Promotion
TYMC	TAMC	TYMC	TAMC	Preparation of Test Strain	Microorganism
	Soybean–Casein Digest Agar/MPN Soybean–Casein Digest Broth ≥100 cfu 30-35°C ≥ 3 days		Soybean–Casein Digest Agar and Soybean–Casein Digest Broth ≥ 100 cfu 30-35°C≥ 3 days	Soybean–Casein Digest Agar or Soybean–Casein Digest Broth 30-35°C 18–24 hours	Staphylococcus aures such as ATCC 6538
	Soybean–Casein Digest Agar/MPN Soybean–Casein Digest Broth ≥ 100 cfu 30-35°C ≥ 3 days		Soybean–Casein Digest Agar and Soybean–Casein Digest Broth ≥ 100 cfu 30-35°C≥ 3 days	Soybean–Casein Digest Agar or Soybean–Casein Digest Broth 30-35°C 18–24 hours	Pseudomonas aeruginosa such as ATCC 9027
	Soybean–Casein Digest Agar/MPN Soybean–Casein Digest Broth ≥ 100 cfu 30-35°C ≥ 3 days		Soybean–Casein Digest Agar and Soybean–Casein Digest Broth ≥ 100 cfu 30-35°C≥ 3 days	Soybean–Casein Digest Agar or Soybean–Casein Digest Broth 30-35°C 18–24 hours	Bacillus subtilis such as ATCC 6633
Sabouraud Dextrose Agar ≥ 100 cfu 20-25°C ≥ 5 days	Soybean–Casein Digest Agar ≥ 100 cfu 30-35°C ≥ 5 days MPN: not applicable	Sabouraud Dextrose Agar ≥ 100 cfu 20-25°C ≥ 5 days	Soybean–Casein Digest Agar ≥ 100 cfu 30-35°C≥ 5	Sabouraud Dextrose Agar or Sabouraud Dextrose Broth 20-25°C 2-3 days	Candida Albicans such as ATCC 10231
Sabouraud Dextrose Agar ≥ 100 cfu 20-25°C ≥ 5 days	Soybean–Casein Digest Agar ≥ 100 cfu 30-35°C ≥ 5 days MPN: not applicable	Sabouraud Dextrose Agar ≥ 100 cfu 20-25°C ≥ 5 days	Soybean–Casein Digest Agar ≥ 100 cfu 30-35°C≥ 5	Sabouraud Dextrose Agar or Potato–Dextrose Agar 20-25°C 5-7 days, or until good sporulation is achieved	Aspergillus brasiliensis such as ATCC 16404

Third step: counting
There are several methods for counting, including:

Colony counting methods on plates
Filtration
And MPN (most probable number) methods (Figure 1, Table 5).

The MPN method is less accurate than other methods, although it may be the most appropriate method for specific product with low microbial loads.

Figure 1. Scheme of MPN method

Identification test for Escherichia coli
Dilute the sample 10 times that equal to 1 g or 1 cc of the product, then transfer it to the soy bean casein digest broth and mix, incubate for 18-24 hours at 35-30°C.
Subculture: After incubation, 1 mL of the soy bean casein digest broth was transferred to 100 mL of MacConkey broth (incubation at 42-44°C, 24-48 h); finally the sample was transferred from MacConkey broth to the MacConkey agar medium (incubation 18–72 h, 30–35°C).
The product is acceptable when no colony growth is observed and the identification test is negative.

Identification Test for Salmonella
Prepare the sample which is equal to 10 g or 10 cc of the product. After transferring the sample to the soy bean casein digest broth medium and mixing, incubation perform for 24 to 18 hours at 30-35°C.
0.1 mL of soy bean casein broth was transferred to 10 mL of Rappoport Vassiliadis Salmonella Enrichment Broth medium and incubated for 18-24 h at 30-35°C. Then transfer to Xylose lysine Deoxycholate Agar medium and incubate at 30-35°C for 18-72 hours.
The presence of Salmonella is characterized by the growth of red colonies with or without black centers, which is confirmed by complementary identification tests. The product is acceptable when no colony growth is observed and the complementary identification tests is negative.

Identification Test for Pseudomonas aeruginosa
Dilute the sample 10 times that at least equal to 1 g of the product. Use 10 cc or any other amounts which is equal to 1 g or 1 cc of the product and mix with appropriate volume of soy bean casein digest broth medium.
When the transdermal patch is tested, a volume of the sample is filtered through a sterile membrane filter to make it equivalent to one transdermal patch and then transferred to 100 mL of soy bean casein digest broth medium and Incubate for 24-28 hours at 30-35°C.
Subculture: transferred to the Citrimide Agar media and incubated for 18-72 hours at 30-35°C.
Colony growth indicates the possibility of Pseudomonas aeruginosa presence and is confirmed by complementary identification tests. The product is acceptable when no colony growth is observed and the complementary identification tests is negative.

Identification test for Staphylococcus aureus
As same as identification test for Pseudomonas aeruginosa but the Staphylococcus aureus is sub cultured in mannitol salt agar medium.
The growth of yellow or white colonies surrounded by yellow halo indicates the possibility of S. aureus presence and is confirmed by complementary identification tests. The product is acceptable when no colony growth is observed and the complementary identification tests is negative.

Identification test for Clostridia
The sample was diluted for 10 times (at least up to 20 cc) which at least equaled to 2 g or 2 cc of the product, it was then divided into two part. One of these parts was heated to 80°C for 10 minutes and cooled rapidly while the other part should not heated.
Subculture: 10 cc or the amount which was equals to 1 g or 1 cc of the product (for each of the two parts) was mixed with suitable amount of refined Clostridia culture medium and is incubated under anaerobic conditions at 35-35°C for 48-72 hours. After incubation for each of two samples, subculture were done in Columbia Agar medium and then incubated under anaerobic conditions at 30-35°C for 48-72 hours.
Anaerobic growth of rod bacteria (with or without endospores) and negative catalase test, indicated the presence of Clostridia, and were confirmed by complementary identification tests. The product was acceptable when no colony growth was observed and the complementary identification tests were negative.

Identification Test for Candida albicans
10 cc or any other amounts which was equal to 1 g or 1 cc or more of the product was mixed with appropriate volume of sabouraud dextrose broth and incubated for 3-5 days at 30-35°C.
Subculture: subculture was done on sabouraud dextrose agar medium and incubated for 4-4 hours at 35-30°C.
The growth of white colonies can indicate the presence of Candida albicans and is confirmed by complementary identification tests. The product is acceptable when no colony growth is observed and the complementary identification tests is negative
Table 8 describes the growth promoting, inhibitory, and indicative properties of media.

Table 6. Aerobic microorganisms in identification test (NCTC: National Collection of Type Cultures)

NCTC	Aerobic microorganisms
Such as ATCC 6538	Staphylococcus aures
Such as ATCC 9027	Pseudomonas aeruginosa
Such as ATCC 8739	Escherichia coli
Such as ATCC 14028	Salmonella enterica subsp. enterica serovar Typhimurium or,
Such as NCTC 6017	as an alternative, Salmonella enterica subsp. enterica serovar Abony
Such as ATCC 10231	Candida albicans

Table 7. Interpretation of Results

	Results for Each Quantity of Product
Probable Number of Bacteria per g or mL of Product	0.001 g or 0.001 mL	0.01 g or 0.01 mL	0.1 g or0.1 mL
more than 10³	+	+	+
less than 10³ and more than 10²	-	+	+
less than 10²and more than 10	-	-	+
less than 10	-	-	-

Table 8. Growth promoting, inhibitory, and indicative properties of media

Test Strains	Property	Test/Medium
Test for bile tolerant Gram negative bacteria
*E. coli*	Growth promoting	Enterobacteria Enrichment Broth Mossel
*P. aeruginosa*
*S. aureus*	Inhibitory
*E. coli*	Growth promoting+ Indicative	Violet Red Bile Glucose Agar
*P. aeruginosa*
*Escherichia coli* Tet for
*E. coli*	Growth promoting	MacConkey Broth
*S. aureus*	Inhibitory
*E. coli*	Growth promoting+ Indicative	MacConkey Agar
Test for salmonella
*Salmonella enterica subsp. enterica serovar Typhimurium or*	Growth promoting	Rappaport Vassiliadis Salmonella Enrichment Broth
*Salmonella enterica subsp. enterica serovar Abony*
*S. aureus*	Inhibitory
*Salmonella enterica subsp. enterica serovar Typhimurium or*	Growth promoting+ Indicative	Xylose Lysine Deoxycholate Agar
*Salmonella enterica subsp. enterica serovar Abony*
*Test for Pseudomonas aeruginosa*
*P. aeruginosa*	Growth promoting	Cetrimide Agar
*E. coli*	Inhibitory
*Staphylococcus* *aureus* Test for
*S. aureus*	Growth promoting+ Indicative	Mannitol Salt Agar
*E. coli*	Inhibitory
*Test* for Clostridia
*Cl. sporogenes*	Growth promoting	Reinforced Medium for Clostridia
*Cl. sporogenes*	Growth promoting	Columbia Agar
*Candida* *albicans* Test for
*C. albicans*	Growth promoting	Sabouraud Dextrose Broth
*C. albicans*	Growth promoting+ Indicative	Sabouraud Dextrose Agar

Conclusion

Non-sterile products should be within the permissible range of microbial content in order to maintain the safety, efficacy and quality of the product. Objectionable microorganisms can be counted and identified using methods described in detail in chapters <61> and <62> of USP in relation to sample preparation, counting and identification. However, the pharmaceutical industry must also pay attention to such microorganisms that are not listed on the USP, but could contaminate the product depending on their production conditions. Products with low aqueous activity may be resistant to bacterial growth, but contaminating microorganisms may survive and have the potential risk to contaminate the product. Adhering to the principles of cGMP lead to preventing contamination during production which causes the producing of the products that have high quality and stability, as a result greater satisfaction and confidence from consumers will be achieved. Due to the fact that there are very few studies on the evaluation of non-sterile products in terms of microbial content in Iran, there is a greater need for such studies domestically.

Conflicts of Interest

Authors declared no conflict of interests.

Type of Study: Systematic Review | Subject: Industrial Microbiology
Received: 2019/09/24 | Accepted: 2020/01/2 | ePublished: 2020/01/10

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