Current HPLC Methods for Assay of Nano Drug Delivery Systems

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Current HPLC Methods for Assay of Nano Drug Delivery Systems

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BENTHAM SCIENCE

Current Topics in Medicinal Chemistry

Serife Evrim Kepekci Tekkeli* and Mustafa Volkan Kiziltas Department of Analytical Chemistry, Faculty of Pharmacy, Bezmialem Vakif University, Istanbul, Turkey

ARTICLE HISTORY Received: September 14, 2015 Revised: August 01, 2016 Accepted: September, 14, 2016 DOI: 10.2174/15680266166661612221 12305

Abstract: In nano drug formulations the mechanism of release is a critical process to recognize controlled and targeted drug delivery systems. In order to gain high bioavailability and specificity from the drug to reach its therapeutic goal, the active substance must be loaded into the nanoparticles efficiently. Therefore, the amount in biological fluids or tissues and the remaining amount in nano carriers are very important parameters to understand the potential of the nano drug delivery systems. For this aim, suitable and validated quantitation methods are required to determine released drug concentrations from nano pharmaceutical formulations. HPLC (High Performance Liquid Chromatography) is one of the most common techniques used for determination of released drug content out of nano drug formulations, in different physical conditions, over different periods of time. Since there are many types of HPLC methods depending on detector and column types, it is a challenge for the researchers to choose a suitable method that is simple, fast and validated HPLC techniques for their nano drug delivery systems. This review’s goal is to compare HPLC methods that are currently used in different nano drug delivery systems in order to provide detailed and useful information for researchers.

Keywords: HPLC, Nano Drug delivery systems, Nano carriers, Drug release, Biological fluids, Pharmaceutical preparations. 1. INTRODUCTION Currently, nano drug formulations are preferred because of their improved bioavailability, solubility and stability, reduced toxicity and especially for their ability to be targeted to specific sites in the body. Based on these advantages, because of their efficiency in being delivered to nano drug formulations are desirable for many applications, including: cancer therapies, imaging agents, vaccines, nutraceuticals, and cosmetics [1]. The development of nano drug delivery systems is, therefore, an important field of nano technology. Nano drug delivery systems can be either lipid or polymer-based. Depending on the physiochemical properties of a drug, the pharmacokinetics and bioavailability can differ in these two types of delivery systems. The first developed nano drugs were non targeted lipid nanoparticles but now there are a respectable number of lipidic and polymeric targeted nano drugs being marketed and used in clinical trials [2]. High performance liquid chromatography (HPLC) is one of the widely used analytical techniques for analysis in biological systems and drug preparations. Due to its ability to detect low amounts in samples as semi, micro and trace levels, it is also mostly preferred for nano drug formulation analyses. In HPLC, as in all chromatographic methods, *Address correspondence to this author at the Department of Analytical Chemistry, Faculty of Pharmacy, Bezmialem Vakif University, P.O. Box: 34093, Istanbul, Turkey; Tel/Fax: ++90-535-826-0871; E-mail: [email protected] 1873-5294/17 $58.00+.00

components of a mixture are partitioned between an adsorbent (the stationary phase) and a solvent (the mobile phase). These phases vary based on the structure of the nano carrier and also drug substances. The detection type differs depending on the structure of the analyte and the amount in the formulation or biological matrices. The goal of developing new drug formulations is to enhance bioavailability and to reduce side effects. To evaluate the therapeutic potential of a drug preparation analytical procedures are required. The amount of drug in plasma, serum, urine or other biological fluid or tissue provides information about the released and therapeutic dose. Correspondingly, our goal in the writing of the manuscript is to review the liquid chromatographic analytical methods, in order to direct researchers on the effects of nano preparations, in addition to helping to develop new analytical methods for nano drug systems. 1.1. Analytical Methods 1.1.1. Determination in Natural Nano Carriers A chitosan based nano carrier including a flavonoid called naringenin, which is known for its strong antioxidant activity, was analyzed using reverse phase high performance liquid chromatography (RP-HPLC). Gradient elution was used to get an efficient resolution value using water:acetonitrile as mobile phase. A photodiodarray (PDA) detector was used at λ 240, 280, 360 and 520 nm wavelengths to look at absorption intensities of naringenin. En© 2017 Bentham Science Publishers

Current HPLC Methods for Assay of Nano Drug Delivery Systems

capsulation efficiency was also measured by using this assay [3]. Sirolimus (rapamycin, Rapamune®;) is an immunosuppressant drug, in this study it is coated with nano porous carbon stents. The release of the active substance is measured by HPLC. For in vitro release measurement, drug eluting nano porous carbon stents were dissolved in phosphate buffer saline (PBS) pH 7.2. The samples were collected at different time intervals according to study design. Collected drug contained PBS samples were extracted with 2 mL of chloroform. Drug extracted chloroform was evaporated under nitrogen. These samples were dissolved in mobile phase. A liquid chromatography-tandem mass combination (LC/MS/MS) assay for sirolimus was developed to quantify it in blood. Octadecylsilane (C18) column (250 × 4.60 mm × 5 µm). and mobile phase including acetonitrile:water, 0.1% acetic acid (80 : 20) was used. Chromatographic separations were performed at 38ºC. The flow rate was set to 1 mL/min. Micro mass triple quadrupole mass spectrometer with an ESI+ source was used for mass analysis and detection. Mass spectrometric analysis was achieved in the positive ion mode [4]. In order to increase the oral bioavailability of two antifungal drugs clotrimazole and econazole a synthetic polymeric (PLG) and a natural polymeric (alginate stabilized with chitosan) nano formulations were developed. To compare the efficacy of the nano carriers the amount of the released drugs were investigated in mice. Pharmacokinetic data were found according to the measurements and the area under the pharmacokinetic curve was calculated to find out the bioavailability of the drugs. The drugs were analyzed by an HPLC system with UV detector at λ 254 nm. In an isocratic program dibasic potassium phosphate buffer:methanol (1:9) were used as mobile phase with 1.5 mL/min flow rate. C18 column, 250 × 4.6 mm × 5µm particle size, was used. The method was found to work equally well for clotrimazole and econazole (analytical sensitivity = 0.2μg/mLg/mL for each drug) [5]. 1.1.2. Determination in Semi-synthetic Nano Carriers Ascorbic acid (Vitamin C), was successfully loaded into solid lipid nanoparticules (SLN) by hot homogenization and efficient transportation of ascorbic acid was delivered to cancer cells. These nanoparticles have a small size, good physicochemical stability, offer controlled release of drugs, and maintain release for a long period of time. The in vitro released ascorbic acid level is measured by HPLC using UV detector. The mobile phase consisted of methanol:water:phosphate buffer (2:78:20) with a flow rate of 1 mL/min. Samples were eluted on a C18 column (25 cm × 2.5 cm, 5 µm) at room temperature. The amount of ascorbic acid loaded into SLN was monitored at a wavelength of λ 254 nm. The retention time was about 4.8 min [6]. Arteether, an artemisinin derivative, is a life saving drug for multiple drug resistant malaria. SLN by high pressure homogenization technique was used to carry the drug in rats. The bioavailability was evaluated by HPLC. A binary gradient pump was used, the detection was set at λ 214 nm with an injection volume of 20 µL, the mobile phase was acetonitrile: water (70:30) with a flow rate 1 mL/min [7].

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Development and characterization of two nano-structured systems for topical application of flavanones isolated from Eysenhardtia platycarpa was performed in a study in 2014. The in vitro release of these preparations was evaluated by RP-HPLC combined with UV detection at λ 290 nm. The mobile phase was acetonitrile:water (80:20) with a flow rate 1 mL/min. The column size was 250 mm x 20 mm with a particule size of 5 µm. Before analysis the drug was extracted into an ethanol:water medium. The free drug in dispersion medium was then measured [8]. Curcumin has a high potential to be used as a neuroprotective and anti-cancer agent, however it is easily eliminated from the body. To improve its penetration and circulation into the different parts of the body and brain, nano particulation methods have been used. Curcumin loaded chitosan-graft-poly (N-vinyl caprolactam) nano carrier containing gold nanoparticles (Au-CRC-TRC-NPs) were analyzed with RP-HPLC (C18 column (150 mm x 4.60 mm x 5 μm) in 1±0.2 min. UV-vis detector was used at λ 429 nm. The amount in plasma and organ tissues were assayed. The sample was extracted by using ethanol and then HPLC analyses of supernatant was done [9]. Efavirenz is a nonnucleoside inhibitor used in the treatment of HIV. Since it is poorly soluble in water, SLN drug delivery systems can be applied to develop its bioavailability. The efavirenz content of SLN formulations was analyzed with RP-HPLC method. C18 column (4.6mm × 5
m) was used in an isocratic assay with a mobile phase of acetonitrile:pH 7.4 ammonium acetate buffer (50: 50) at 1.0 mL/min. The detection wavelength was set at λ 246 nm [10]. In vitro release kinetics of antituberculosis drugs (moxifloxacin and rifampicin) loaded nanoparticle system, in which gelatin and polybutyl cyanoacrylate (PBCA) were used as nano carriers. The release capacity was studied with a RP-HPLC assay. The drug concentrations were determined by the set of data obtained from the separation carried on C18, 5
m column with a mobile phase of methanol: 0.3% v/v trimethylamine,0.02M PBS (pH 3.0) (40:60) at a flow rate of 0.9 mL/min. Detection was set at the wavelength of λ 295 nm [11]. Haloperidol is used to cure some certain psychiatric conditions like schizophrenia, manic states, medication-induced psychosis and neurological disorders. Enhanced intranasal delivery of haloperidol to brain was developed by the use of SLNs. In vitro haloperidol release study was done by the RPHPLC system consisting of a C18 column (250 mm × 4.6 mm × particle size 5 µm) and a mobile phase of potassium dihydrogen phosphate:acetonitrile: triethylamine (10:90:0.1) at a flow rate of 2 mL/min. The eluent detection was monitored at 230 nm [12]. Drug conjugation of hydrophobic and hydrophilic drugs on the same drug delivery system (lipid–polymer hybrid nanoparticle) study was reported. Paclitaxel and cisplatin was used as a hydrophobic and hydrophilic drug, respectively. The paclitaxel concentration was determined by RPHPLC with a C18 column (4.6 mm ×150 mm) and a mobile phase of acetonitrile:water (50:50) [13]. In another study, it is reported that biodegradable nanoparticles based on recombinant human gelatin modified

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with alphatocopheryl succinatethe was developed. The nanoparticles encapsulated 17-AAG (17-allylamino-17demethoxygeldanamycin), a small molecular anticancer drug targeting heat shock protein. The loading efficiency, of the nano carrier was determined using HPLC. HPLC combined with a UV detector and a C18 column. The mobile phase was (31:43:26) acetonitrile: methanol:water, and the samples were eluted in an isocratic condition of the mobile phase at a flow rate of 1 mL/min and detected at λ 254 nm [14]. 1.1.3. Determination in Synthetic Nano Carriers In another RP-HPLC method, an aqueous mobile phase was used with %0.1 formic acid and acetonitrile at a flow rate of 0.4 mL/min to measure the released amount of nonsteroidal anti-inflammatory drug substance indomethacine in zirconia and poly(ε-caprolactone) (ZrO2/PCL) carrier. The primary aim of this preparation is to provide implantable therapeutic systems for bone or tooth repair, which can also release drugs and reduce inflammatory or infectious side effects of implants. The releasing process was conducted in vitro by using 2.0 mL Dulbecco's Phosphate Buffer Saline (DPBS) simulated body fluid at 37ºC under continuous stirring. Ultra-violet absorption detector was used at λ 320 nm [15]. In another HPLC method, insulin loaded pH sensitive nanohydrogels were investigated according to their insulin releasing ability. In this study, the performance of pH sensitive nanohydrogel, as insulin delivery system, with good smart-releasing behavior for oral administration of insulin was investigated. The in vitro release of insulin from nanohydrogel was performed at 37ºC at two different pH’s (pH 1.2 and 6.8). Samples were collected as different times and HPLC was used for analysis of samples using mobile phase of water:acetonitrile (65:35). The UV detection was set at λ 214 nm. The total run time for the assay was 15 min and the retention time of insulin was 2.56 min. The use of HPLC, help to determine that insulin was best delivered using nanohydrogel [16]. With a different HPLC method in literature, the permeability of clarithromycin poly(lactic-co-glycolic acid) (PLGA) nanoparticles using single-pass intestinal perfusion technique in rats was determined. Clarithromycin nanoparticles were prepared by nano-precipitation according to the modified emulsion solvent diffusion technique and evaluated for their physiochemical characteristics. Phenol red (0.7 mM) and metoprolol (0.07 mM) were added to the solution in all experiments as non-absorbable marker and internal standard, respectively. An indirect quantitation was applied, and the phenol red concentration was measured in samples. A previously developed HPLC method was used for phenol red analysis [17]. The HPLC conditions consisted of a C18 column and a mobile phase of 55% methanol and 45% 0.05 mol/L KH2PO4 at pH 2.6. Flow rate was 1 mL/min the retention time of phenol red was 3 min. The detection was performed using UV-vis detector set at λ 430 nm. Effective permeability of curcumin to intestinal tissue was evaluated by permeability coefficients, that were calculated, after correcting the steady-state outlet concentrations for water flux, based on the ratio of inlet and outlet concentrations of an unabsorbable marker, phenol red [18].

Tekkeli and Kiziltas

A novel cyclodextrin (CD)-based nanoparticle drug delivery system was assembled and characterized for the therapy of folate receptor-positive [FR(+)] cancer. Water-soluble folic acid (FA)- conjugated CD carriers were successfully synthesized and their structures were confirmed by 1D/2D nuclear magnetic resonance (NMR), matrix-assisted laser desorption ionization time-of-flight mass spectrometer (MALDI-TOF-MS) and HPLC. HPLC analyses were carried out using binary pumps, a PDA and an electrospray light scattering detector (ELSD) detector. The HPLC was equipped with a C18 column with a particle size of 3.5 µm and column size of 100 mm length, 4.6 mm diameter. The mobile phase consisted of acetonitrile and water with a flow rate 1.0 mL/min, while the HPLC pressure was controlled between 260-290 bars [19]. In this study chromatography was used for separation only, the spectra provided qualitative data. To investigate the efficacy of a magnetic carrier drug delivery system, paclitaxel was used as loaded drug and analyzed by HPLC. Paclitaxel-loaded carrier was synthesized by octadecyl-quatemized carboxymethyl chitosan, Fe3O4, cholesterol and paclitaxel at a weight ratio of 4:2:3:1. The synthesized suspension was purified by centrifugation at 15 000 rpm at 4ºC for 3 min. The paclitaxel in supernatant was evaluated by HPLC. The loading and encapsulation efficiency was calculated by using a previously developed method [20]. The mobile phase was acetonitrile: methanol: water, stationary phase was a C18 column, the analysis was conducted at a rate of 1 mL/min and with UV-vis detection. The release process was conducted in vitro. Drug-loaded magnetic carrier was contained in a dialysis bag and dispersed in 20 mL PBS (pH 7.4) containing 1% Tween 80 at 37±0.5ºC [21]. Nano formulations are also used for nutraceuticals. Some substances, which have health benefits, are being isolated from foods or some natural sources in order to use them in nutraceuticals. However most of these components have low bioavailability and poor solubility. Nano formulation scientists are currently working on to improve their bioavailability and bioactivity. The release from raw foods was enhanced in this study by using nano carriers. The aim of the cited study was to evaluate whether different particle processing methods might affect the lipid-lowering activity of Dioscorea pseudojaponica, and to investigate whether it could be used as a potential functional food for prevention of atherogenesis in rabbits. RP- HPLC was used to measure serum lipid levels. Prior to HPLC analysis, the external standard and analyte were dissolved in methanol and filtered. A C18 column and UV detector at λ 205 nm was used. The mobile phase was methanol:water (95:5) at a consistent flow rate of 1 mL/ min [22] (Table 1). Stevioside is a FDA approved nontoxic natural noncaloric sweetener. A nano formulation using Pluronic-F-68 copolymer based polyactic acid (PLA) nanoparticles by nanoprecipitation was developed to overcome intestinal malabsorption and to enhance its bioavailability. The amount of analyte in a nanocapsule was determined by HPLC. By using this analytical method the preparation was standardized. The powdered preparation was dissolved in 1.0 mL (80:20) acetonitrile:water solution. This solution was directly

Current HPLC Methods for Assay of Nano Drug Delivery Systems

Table 1.

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The list of analytical conditions of the nano drug formulations.

Analyte

Sample Matrix

Nano Carrier Type

Stationary Phase

Mobile Phase

Flow Rate mL/min

Detection Type

Naringenin [3]

nanoformulation

chitosan based

C18

water:acetonitrile

0.8

PDA

Rapamycin [4]

PBS (in vitro release)

nano porous carbon stent

C18

water: acetonitrile

1

MS

Clotrimazole Econazole [5]

mice plasma

natural polymeric (alginate stabilized with chitosan)

C18

acetonitrile:sodium sulphate buffer

1

UV

ascorbic acid [6]

nanoformulation

SLN

C18

methanol:water:phosphate buffer

1

UV

Arteether [7]

rat plasma

SLN

C18

water: acetonitrile

1

UV

1

UV

lipid–polymer hybrid nanoparticles

nucleosil column

water: acetonitrile

human skin

5,7-Dihydroxy-6-methyl-8prenylflavanone 5,7-dihydroxy-6-methyl-8prenyl-4-methoxy- flavanone 5,7-dihydroxy-6prenylflavanone 5-hydroxy-7-methoxy-6Prenylflavanone [8] Curcumin [9]

human plasma and chitosan-graft-poly (N-vinyl tissues caprolactam) nanocarrier containing gold nanoparticles

C18

acetonitrile:acetic acid

1

UV-vis

Efavirenz [10]

nanoformulation

SLN

C18

dibasic potassium phosphate buffer:methanol

1.5

UV

Moxifloxacin Rifampicin [11]

in media of different pH and enzymatic degradation of gelatin NPs

gelatin and PBCA

C18

acetonitrile:methanol: ammonium acetate

0.75

UV

Haloperidol [12]

SLN ethanol and chloroform(1:1) solution

C18

water 0.1% (v/v) TFA :acetonitrile 0.1% (v/v) TFA

1

UV

Paclitaxel Cisplatin [13]

PBS (in vitro release)

lipid–polymer hybrid

C18

water: acetonitrile

0.9

MS

recombinant human gelatin modified with alphatocopheryl succinatethe

C18

acetonitrile: phosphate buffer

-

UV

C18

formic acid: acetonitrile

0.4

UV

-

water: acetonitrile

-

UV

17-AAG (17- allylamino-17PBS (in vitro demethoxygeldanamycin)-[14] release)

Indomethacine [15]

artificial saliva (in zirconia and poly(εvitro release) caprolactone) (ZrO2/PCL)

Insulin [16]

PBS (in vitro release)

Clarithromycin [17,18]

rat intestine tissue PLGA

C18

methanol:phosphate buffer

1

UV-vis

cyclodextrin based nanoparticle [19]

cyclodextrin ----(qualitative study, analyte directly investigated)

C18

water: acetonitrile

1

PDA, ELSD

Paclitaxel [20,21]

PBS (in vitro release)

acetonitrile:methanol:water

1

UV-vis

pH sensitive nanohydrogel

C18 octadecyl-quatemized carboxymethyl chitosan, including Fe3O4

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Tekkeli and Kiziltas

(Table 1) contd…. Analyte

Sample Matrix

Nano Carrier Type

Dioscorea pseudojaponica [22] rabbits serum

Stationary Phase

Mobile Phase

Flow Rate Detection mL/min Type

C18

methanol:water

1

UV

Amino column

water: acetonitrile

1

PDA

Stevioside [23]

nanocapsule

pluronic-F-68 copolymer based PLA

Prednisolone [24]

nanoformulation

PAA modified with fluoren- C18 ylmethoxy carbonyl, dimethylamino-1naphthalenesulfonyl and cholesteryl group

acetonitrile:water

1

UV

Propofol [24]

nanoformulation

PAA modified with fluoren- C18 ylmethoxy carbonyl, dimethylamino-1naphthalenesulfonyl and cholesteryl group

methanol:water

1

UV

Griseofulvin [25]

nanoformulation

PAA modified with fluoren- C18 ylmethoxy carbonyl, dimethylamino-1naphthalenesulfonyl and cholesteryl group

acetonitrile:45 mM potassium dihydrogen phosphate buffer

1

UV

Insulin [26]

nanoformulation

Poly (PEGDMA-MAA)

C18

methanol:water

1

UV

Paclitaxel [27]

PBS (in vitro release)

chitosan and glyceryl monooleate

C18

acetonitrile:methanol:water

1

UV

Insulin [28]

PLGA in an organic/water (O/W) interface

C18

water:acetic acid

1

UV

Paclitaxel [29]

human cervix carcinoma cells

PEGylated PLGA

C18

acetonitril:ammonium acetate buffer

1

UV

Haloperidol [30]

nanoformulation

PLGA

C18

potassium dihydrogen phosphate:acetonitrile:triethylamine

2

UV

Paclitaxel [31]

nanoformulation

folate-functionalized hybrid polymeric nanoparticles

C18

acetonitril:water

1

UV

injected to HPLC. The PDA detector was used at λ 205 nm. An amino column (150 mm × 4.6 mm, 5 µm size) was used with a flow rate of 1 mL/min. The calibration curve was constructed using different amounts of pure stevioside (50–500 g/mL) [23]. The use of nano self-assemblies, formed by polyallylamine (PAA)+ modified with fluorenylmethoxy carbonyl, dimethylamino-1-naphthalenesulfonyl and cholesteryl group for oral hydrophobic drug delivery was investigated. Propofol, griseofulvin and prednisolone were loaded into amphiphilic PAAs. In vitro drug release and formulation stability was analyzed by HPLC. For quantitation, a C18 150 mm×4.6 mm×3.5 μm column with the mobile phase (36:64) acetonitrile:water and a flow rate of 1 mL/min was used. The prednisolone peak eluted at 3 min and was detected at λ max 243 nm. For quantification of propofol, a C18 250 mm × 46 mm × 5 μm column was used with the flow rate of 1 mL/min

(80:20) methanol:water in an isocratic elution. In 7 min the analyse was completed at λ 229 nm [24]. For quantification of griseofulvin Trimaille’s method [25] was used. C18 250 mm×46 mm×5 μm column is used and the peak (9.5 min) was detected at λ 293 nm. The mobile phase was (45:55) acetonitrile:45 mM potassium dihydrogen phosphate buffer (adjusted to pH 3 with orthophosphoric acid). Poly (PEGDMA-MAA) copolymeric micro and nanoparticles for oral insulin delivery was evaluated by an in vitro insulin release study. Various insulin loaded particles were performed by simulating the gastrointestinal tract conditions using HPLC. Insulin loaded polymeric nanoparticles were freeze dried and weighed. Citrate–phosphate buffer solutions at pH 2.5, after 90 min, pH of the medium was changed to 7.4 by adding fresh phosphate buffer and samples were collected. Supernatants were collected after each centrifugation were then analyzed by RP-HPLC with C18 column and mo-

Current HPLC Methods for Assay of Nano Drug Delivery Systems

bile phase consisted of acetonitrile and sodium sulphate buffer of pH 2.3 in the ratio of (24:76) with a flow rate of 1.0 mL/min, UV detector at λ 214 nm [26]. To provide sustained release of paclitaxel nanoformulation consisting of chitosan and glyceryl monooleate was developed. To determine its therapeutic potential in vitro release and cellular uptake were determined using HPLC with C18 column (4.6 mm, 250 mm, 5 μm; with a mobile phase consisting of acetonitrile: methanol: 0.1 M ammonium acetate (48.5:16.5:35) at a flow rate of 0.75 mL/min. The drug was detected at λ 227 nm. Before chromatographic process the drug was extracted in PBS (pH 7.4) [27]. The stability of insulin in PLGA microspheres was studied by application of a polymeric complex between a therapeutic peptide and chargeable polymer. The nano sized (100400 nm) complex was formed with chondroitin sulfate as a polymer additive for the formation of ionic complex with insulin. The stability studies were conducted via RP-HPLC. Binary gradient study was done with a mobile phase of solution A: water 0.1% trifluoroacetic acid (TFA) and solution B: acetonitrile with 0.1% TFA with a flow rate of 1.0 mL/min. C18 (15cm×0.46 cm, 300 Å particle size) column was used and detection wavelength was set at λ 276 nm [28]. Cremophor® EL nanoparticles was used in the delivery of paclitaxel through parenteral route. Since it has some side effects such as hypersentivity, nephrotoxicity and neurotoxicity, the study was designed to improve paclitaxel loaded Cremophor® EL-free nanoparticles for the intravenous administration. Paclitaxel -loaded PEGylated PLGA-based were used instead of Cremophor® EL-free nanoparticles. The efficiency of the drug loading was analyzed with RP-HPLC system. C18 column was used with a mobile phase acetonitrile:water (70:30) at 1.0 mL/min. Detection wavelength was λ 227 nm [29]. As a biodegradable polymer, PLGA was used in longterm drug delivery studies as a carrier. One of the most common anti-psychotic agent, Haloperidol was loaded into PLGA nano particles to study on controlled drug release parameters. Haloperidol content of PLGA nano particles was carried out by RP-HPLC (C18 column 5µm, 4.6 ×150mm). The mobile phase composition was 38% acetonitrile and 62% 10mM, pH 4.8 ammonium acetate solution. UV detection was done at λ 254 nm [30]. As a low water soluble tumor targeting drug, paclitaxel different nanoparticle carrier studies was prepared to improve its penetration. Folate-functionalized hybrid polymeric nanoparticles is one of the studies, the efficacy of the carrier was carried out with an HPLC method to analyse loaded amount of the drug. The samples carried out with a mobile phase consisted of acetonitrile:water (70:30) at 1.0 mL/min. UV detection of paclitaxel was achieved at λ 227 nm [31].

Current Topics in Medicinal Chemistry, 2017, Vol. 17, No. 13

As demonstrated from the literature survey, sensitive analytical methods have always been required for the analysis of nanoformulations. In order to determine the loaded amount of a drug or the released amount, generally chromatographic methods have been applied. According to the literature survey it is clearly understood that HPLC is mostly preferred method for assay of nano formulations with its various detection types, divergent column applications in terms of chemical structure of analyte and other components of matrices. Especially for non volatile and biologically soluble compounds HPLC is the only choice for sensitive and accurate quantitative analyses. Generally RP HPLC methods were used for polymeric or lipidic nano carriers. The use of a non polar C18 column, along with an aqueous mobile phase resulted in good resolution values of improved separation of drugs from different matrices. Only one study in the literature search describes an analytical procedure using a polar amino column [16]. However it is also relatively RP HPLC based on the polarity differences of the stationary and mobile phases. As a result, it is exactly seen that HPLC is an efficient technique to evaluate the efficiency of nano drug formulations. CONFLICT OF INTEREST The authors confirm that this article content has no conflict of interest. ACKNOWLEDGEMENTS Declared none. REFERENCES [1]

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[5]

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[7]

CONCLUSION Nano formulations are mostly used in the development of anti-cancer agents resulting in fewer adverse side effects. The other goal is to develop these formulations based on their improved bioavailability to biological systems for antiinflammatory drugs, nutraceuticals, antibiotics, antipsychotic agents etc.

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