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Agilent 1290 Infinity LC with AgilentPoroshell columns for simultaneousdetermination of eight organic UV filters in under two minutes
Levels of UV filters in personal care products are regulated by the FDA and European
Pharmacopeia (EP). Liquid chromatographic (LC) methods are widely accepted analyt-
ical techniques for the qualitative and quantitative analysis of these UV filters. Most
of these traditional LC methods require about 2550 minutes. In this Application Note,
the Agilent 1290 Infinity LC, in combination with Agilent Poroshell columns, were
used for development of a short, sensitive, robust and well resolved separation of
eight FDA/EP approved active UV filter ingredients in 99 seconds. Standard deviation
(SD) and relative standard deviation (RSD) values of retention time for replicate injec-
tions confirmed the excellent performance of the Agilent 1290 Infinity Binary Pump.
Exceptional performance of the Agilent 1290 Infinity Diode Array Detector was estab-
lished by minimum area RSD values and a wide linear range with standard organic UV
filters in amounts from 0.25 ng to 200 ng on-column. In addition, the method was
effectively used to identify active UV filters extracted from six international personal
Agilent Technologies India Pvt. Ltd.
Agilent Technologies, Inc.
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min0 0.5 1 1.5 2
IntroductionThe wavelength of UV rays liesbetween X-rays and visible light (~ 10 400 nm)1. Production ofmelanin, a pigment that causes thedarkening of skin, is a natural defenseof the human body against UV radia-tion. Melanin absorbs UV radiation anddissipates the energy as harmless heat,though the response to UV radiationand production of melanin pigmentationdepends on skin color and other geneticfactors 2, 3. The intensity of UV radiationand length of exposure are the mainparameters involved in sunburn, irre-spective of skin tone and ability of theskin to produce melanin. The majorclassifications of UV light are presentedin Table 14.
Sunscreens protecting the skin againstsunburn contain one or more of the fol-lowing types of active ingredients:
Organic chemicals that absorb UV light.
Inorganic particulates that reflect, scatter, and absorb UV light.
Organic particulates that have all theabove features.
Organic UV filters are usually aromaticcompounds conjugated with carbonylgroups. The FDA has approved sevenUV-A filter compounds and nine UV-Bfilter compounds for sunscreen formu-lations in the United States, while theEuropean Commission has approvedthe use of ten additional UV filters inEuropean countries5.
Though several approved UV filters areavailable in the market, extensive useof these UV filters may have several
major concerns. Some sunscreen ingre-dients have been shown to have car-cinogenic properties. Additionally, olderand more widespread sunscreen chemi-cals cannot dissipate the energy of theexcited state as efficiently as melanin,therefore the penetration of these sun-screen ingredients into the lower layersof the skin may increase the amount offree radicals and reactive oxygenspecies6. Therefore, extensive testingof sunscreens is advisable to reveal theefficacy of the ingredients. ThisApplication Note discusses a short LCmethod to separate eight widely usedUV filters within 99 seconds. Cosmeticmanufacturers can adopt this methodto simplify the analysis of sunscreenraw materials and personal care prod-ucts in product development, regulatorycompliance, and quality control toincrease the efficiency of analysis.
ExperimentalInstrument configurationAn Agilent 1290 Infinity LC, controlledby ChemStation (Version B.04.02) andequipped with a binary pump with inte-grated vacuum degasser, autosampler,thermostatted column compartment
and a diode array detector, was used fordata collection. The injection volumewas set to 1 L and the needle washwas enabled using acetonitrile for threeseconds. The sample thermostat wasset at 5 C, while the columns wereoperated at 55 C. The binary pump wasoperated at a flow rate of 1 mL/min.The detector was programmed for threedifferent wavelengths (288, 304 and 358nm) and operated at a sampling acqui-sition rate of 80 Hz (response time0.062 seconds, >0.003 min). An AgilentPoroshell 120 EC-C18 column (75 mm 2.1 mm, 2.7 m) was used for the chro-matographic separation.
Chemicals and standardsAll eight UV filter standards and aceticacid (mobile phase modifier) were pur-chased from Aldrich (India). Super gra-dient grade acetonitrile (ACN) was pur-chased from Lab-Scan (Bangkok,Thailand). HPLC grade water was fresh-ly taken from a Milli-Q water purifica-tion system. Six different brands ofinternational sunscreen formulationswere purchased locally. The details oforganic UV filter standards used in thisstudy are tabulated in Table 2.
Sl No: Component Wavelength (nm) Effects of over exposure on skin
1 UV-A I 340-400 Can cause tanning but has minimal erythemal effect. Can cause long term damage. Penetrates deeply. it cancontribute to skin cancer via indirect DNA damage.
2 UV-A II 320-340 Slightly erythemal contribution
3 UV-B 290-320 Causes sunburn and is a major contributor to skin cancerdevelopment.
4 UV-C 100-290 Very energetic radiation. Absorbed by the ozone layer.Direct DNA damage
Table 1Major classifications of UV radiation.
Table 2Detailed list of organic UV filter standards used in this study.
Compound number Compound Structure UV Spectra
1 Name: Aminobenzoic acid CAS No: 150-13-0 Mol Formula: C7H7NO2Mol Wt: 137.14 Detection: 288 nm
2 Name: Dioxybenzone CAS No: 131-53-3 Mol Formula: C14H12O4Mol Wt: 244.24 Detection: 288 nm
3 Name: Oxybenzone CAS No: 131-57-7 Mol Formula: C14H12O3Mol Wt: 228.24 Detection: 288 nm
4 Name: 4-Methyl benzylidene camphor CAS No: 36861-47-9 Mol Formula: C18H22O Mol Wt: 254.37 Detection: 304 nm
5 Name: Avobenzone CAS No: 70356-09-1 Mol Formula: C20H22O3Mol Wt: 310.39 Detection: 358 nm
6 Name: Octylmethoxycinnamate CAS No: 5466-77-3 Mol Formula: C18H26O3Mol Wt: 290.397 Detection: 304 nm
7 Name: Octocrylene CAS No: 6197-30-4 Mol Formula: C24H27NO2Mol Wt: 361.48 Detection: 304 nm
8 Name: Octyl salicylate CAS No: 118-60-5 Mol Formula: C15H22O3Mol Wt: 250.33 Detection: 304 nm
NH2nm250 300 350
nm250 300 350
nm250 300 350
nm250 300 350
nm250 300 350
nm250 300 350
nm250 300 350
nm250 300 350
LC parameters Premixed solutions of 0.1% acetic acidin water and acetonitrile in the ratio90:10 (A) and 10:90 (B) were used asmobile phase. The gradient used for thestudy is presented in Table 3. A post runtime of 1 minute was set for column re-equilibration.
equal volumes of 100 ppm standard mixsolution and extracted sample stocksolution. This spiked sample was usedfor the confirmation of peak identity inextracted samples by means of reten-tion time and UV spectra.
ProcedureA blank injection was performed in alltrials to check the chromatographicinterference in the resolution. Standardmix, linearity levels, diluted extractedsamples and spiked diluted extractedsamples were also injected. The reten-tion time of each standard was con-firmed by individual standard injections.
Results and DiscussionLC chromatogram of standardmixtureThe results showed excellent baselineseparation of all eight active sunscreen
ingredients, without chromatographicblank interference. The last peak of thestandard mix (octyl salicylate) eluted at1.62 minutes. A chromatographic rep-resentation of the standard mix is asshown in Figure 1. An unknown peakwas observed at approximately 0.65 minute, which is an impurity pre-sent in the avobenzone standard. Apeak purity check by spectral scanningin the range of 200 to 400 nm revealedthat all eight compounds eluted with-out co-elution of any detectable impu-rities. Three different wavelengthswere selected for detection as themaximum absorbance values vary forindividual components. The peak width(half height), peak symmetry, USP tail-ing factor, and resolution values confirmthe baseline separation of all the stan-dard analytes in 99 seconds using theAgilent Poroshell 120 EC-C18 column(Table 4).
Time (min) B (%)
Table 3Gradient used for experiment.
Standard mixA premixed solution of acetonitrile and0.1% acetic acid in the ratio 50:50 wasused as the diluent. A stock solution ofeach standard was prepared individual-ly at a concentration of 1000 ppm (1000 ng/L). A standard mixture of p-aminobenzoic acid, dioxybenzone,oxybenzone, 4-methy