2
HROBOŇOVÁ AND LOMENOVA
5.0% (pharmaceuticals up to 10%). For skin care products,
the use of D‐panthenol is recommended; for hair car and
nail care products, D‐ and DL‐panthenol are used.4
solvents for preparation of mobile phases, ethanol, and n‐
hexane were purchased from Merck (Germany), and
propan‐2‐ol was from Fisher Scientific. All solvents were
HPLC gradient grade purity. Triethylamine (for synthe-
sis) was purchased from Merck, and trifluoroacetic acid
(HPLC grade) was from Fisher Scientific. Samples, hair
conditioner (I) and hair spray (II), were bought in the
local supermarket. Samples were stored in dark in the
original packages.
Several achiral analytical methods, such as colorimet-
ric5 and spectrofluorimetric,6 supercritical fluid chroma-
tography (SFC),7 gas chromatography (GC), capillary
electrophoresis (CE),8,9 and high‐performance liquid chro-
matography (HPLC)10 methods, have been reported for
determination of panthenol or its derivatives. Separation
of enantiomers is often applied in the field of pharmaceu-
tical analysis and for determination of optical purity of bio-
logically active compounds because of their different
pharmacokinetic and pharmacodynamic effects. For
direct enantiomeric separation of panthenol, SFC and
GC methods were previously published (Table 1). HPLC
is often applied technique for direct separation of enantio-
mers of many biologically active compounds because of
the availability of a large number different types of chiral
stationary phases (CSPs) and separation modes. By previ-
ously published methods, only poor enantioseparations
of panthenol‐related compounds (DL‐pantoic acid, DL‐
pantothenic acid) were obtained by a ligand‐exchange
HPLC (Rs = 0.4).11,12 More efficient separations and sensi-
tive detection were achieved for 3,5‐dinitrophenyl carba-
mate or 3,5‐dinitrobenzoyl ester derivatives of panthenol.
The separations on chiral acrylic polymeric or Pirkle type
of CSPs in normal phase mode were previously published
(Rs = 1.3).11,13 Frequently, HPLC system with one CSP is
sufficient for separation of enantiomers. In many cases
however, co‐eluting or closely eluting impurities from
complex matrices can interfered in chiral separation or
caused insufficient resolution of compounds. Coupling of
achiral stationary phase with needed achiral selectivity
and CSP (the same mobile phase used for both stationary
phases) can yield selectivity for all components in one
chromatographic method.14
2.2 | Sample preparation
SPE on C18 type (Sep‐Pak C18, Waters, USA) and hybrid
type (OASIS HLB, Waters, USA) cartridges was performed
for extraction of panthenol from hair care samples. The car-
tridge was conditioned with 2 cm3 of methanol and 2 cm3 of
water to activate the sorbent. The appropriate volume of
sample (2 cm3) was loaded, and subsequently, the cartridge
was washed with 3 cm3 of water. Analytes were eluted with
2 cm3 of ethanol. The extraction procedure was repeated
three times for each kind of tested sorbent material.
2.3 | HPLC instrumentation and
conditions
The Agilent 1200 HPLC system (Agilent Technologies,
Germany) consisting of a binary solvent delivery pump,
an injection valve, a column oven compatible with tem-
perature up to 85°C, and a diode array detector was used
for separation and detection of analytes. Data acquisition
was performed by Agilent Chemstation software. The
polarimetric detector (Chiralyser IBZ Messtechnik, Ger-
many) was online coupled to diode array detector. The
Lichrospher 100 NH2 (10 mm × 4 mm I. D, 5 μm particle
size; Merck, Germany), Hypersil APS2 (125 mm × 3 mm
I. D, 5 μm particle size; ThermoFisher Scientific, USA),
and Hypersil CPS2 (125 mm × 3 mm I. D, 5 μm particle
size; ThermoFisher Scientific, USA) columns were tested
for achiral separations. The chiral chromatographic col-
umns LUX i‐Amylose‐1 (250 mm × 4.6 mm I. D, 5 μm
particle size, Phenomenex, USA), ChiraDex (250 mm ×
4.6 mm I. D, 5 μm particle size; Merck, Germany), and
CF6‐P (250 mm × 4 mm, 5 μm; Azyp, USA) were used
for enantioseparations. The mobile phases consisted of
n‐hexane and propanol‐2‐ol or ethanol at different vol-
ume ratios and the flow rate was 0.8 cm3 min−1. The col-
umn temperature was maintained at 25°C or 5°C to 45°C
(for thermodynamic study), and the injection volume was
0.02 cm3. UV spectrophotometric detection was per-
formed at 210 nm. The polarimetric detector worked with
the following settings: the light emitting diode (426 nm)
This research is focused on the development fast and
enantioselective HPLC method for the analysis of
panthenol containing hair care products. Solid‐phase
extraction (SPE) and online coupled achiral‐chiral col-
umns system were applied to increase efficiency of sepa-
ration. The compatibility of mobile phases for both
separation systems were selected. The effects of mobile
phase composition and column temperature on resolu-
tion and retention of enantiomers were also investigated.
2 | MATERIALS AND METHODS
2.1 | Chemicals, solvents, samples
DL‐panthenol (99%) and D‐panthenol (99%) were pur-
chased from Alfa Aesar (Fisher Scientific, USA). Organic