High-performance Liquid Chromatography System Research Articles

Comparison of stereochemical structures of cholesterol from different sources by HPLC

ABSTRACT: It is known that only one stereoisomeric form, nat-cholesterol, naturally occurs. Nat-cholesterol and its enantiomer, ent-cholesterol, sometimes show enantiospecific interactions with biological molecules. If cholesterol is naturally found only one form, then the question of “why does cholesterol show an enantiomeric selectivity?” arises. For this purpose, stereoisomer analysis of cholesterol obtained from porcine liver and wool wax were carried out with three different high performance liquid chromatography (HPLC) systems including reversed-phase, reversed-phase with different cyclodextrins as a mobile phase modifier, and chiral. Results from HPLC analysis of both cholesterol samples by permethylated -cyclodextrin and amylose tris-(3,5-dimethylphenylcarbamate) chiral columns showed that there was no stereoisomer of cholesterol present. However reversed-phase HPLC analysis of cholesterol samples from porcine liver carried out with various cyclodextrins as mobile phase modifiers presented a peak which was not observed in the analysis of cholesterol samples from wool wax. On the other hand, different storage conditions of cholesterol samples and addition of cyclodextrins as mobile phase modifiers produced almost identical alterations in chromatograms of fresh samples by reversed-phase HPLC. This could be attributed to catalytic properties of cyclodextrins. Cyclodextrins may not be suitable as a mobile phase modifier in the stereoisomer analysis of cholesterol with high performance liquid chromatography. KEY WORDS: Cholesterol, stereoisomer, chromatography, chiral, cyclodextrin

Determination of Sedative Component in Chinese Medicines by High Resolution Chromatography-Electron Impact-Mass Spectrometry

The sedative compounds in two traditional Chinese medicines were studied. These compounds are present in the medicinal herbs “Zao Ren” and “Lu Lu Tong”. The samples were first extracted by ethanol and pre-treated by ultrasonic wave, and then they were separated and qualitatively analyzed by high-resolution GC-EI-MS and High Performance Liquid Chromatography (HPLC) systems. We have found that the Chinese herb medicines “Zao Ren” contained clonazepam (120 ppm), whereas “Lu Lu Tong” Chinese drug contained estazolam (270 ppm). The Analytical measurements were done by using three different diagnostic indices using the GC-retention time of the GC-EI-MS total –ion–chromatogram; single stage EI-MS analysis identification and from the measurements of the retention time in the HPLC.

Controlled Drug Release through a Plasma Polymerized Tetramethylcyclo-tetrasiloxane Coating Barrier

A plasma polymerized tetramethylcyclo-tetrasiloxane (TMCTS) coating was deposited onto a metallic biomaterial, 316 stainless steel, to control the release rate of drugs, including daunomycin, rapamycin and NPC-15199 (N-(9-fluorenylmethoxy-carbonyl)-leucine), from the substrate surface. The plasma-state polymerized TMCTS thin film was deposited in a vacuum plasma reactor operated at a radio-frequency of 13.56 MHz, and was highly adhesive to the stainless steel, providing a smooth and hard coating layer for drugs coated on the substrate. To investigate the influence of plasma coating thickness on the drug diffusion profile, coatings were deposited at various time lengths from 20 s to 6 min, depending on the type of drug. Atomic force spectroscopy (AFM) was utilized to characterize coating thickness. Drug elution was measured using a spectrophotometer or high-performance liquid chromatography (HPLC) system. The experimental results indicate that plasma polymerized TMCTS can be used as an over-coating to control drug elution at the desired release rate. The drug-release rate was also found to be dependent on the molecular weight of the drug with plasma coating barrier on top of it. The in vitro cytotoxicity test result suggested that the TMCTS plasma coatings did not produce a cytotoxic response to mammalian cells. The non-cytotoxicity of TMCTS coating plus its high thrombo-resistance and biocompatibility are very beneficial to drug-eluting devices that contact blood.

Determination of Benzalkonium Chloride in Nasal Drops by High-Performance Liquid Chromatography

A high-performance liquid chromatography (HPLC) system was used in the reversed phase mode for the determination of benzalkonium chloride (BKC) in nosal drops. A Chromolit RP-18e, 100 x 4.6, (UM6077/035) column was used at 40 °C. The mobile phase, optimized through an experimental design, was a 70:30 (v/v) mixture of 0.057M Na-heksansulphonate potassium, dihydrogen orthophosphate buffer (pH 2.9) and acetonitrile, pumped at a flow rate of 1.75 mL/min at maintaining column temperature at 40 °C. Maximum UV detection was achieved at 215 nm. The method was validated in terms of selectivity, linearity, repeatability, precision and accuracy. The method was successfully applied for the determination of BKC in a pharmaceutical formulation of nasal drop solution without any interference from common excipients and drug substance. All the validation parameters were within the acceptance range, concordant to ICH guidelines.

153 HEPARIN-BINDING OVIDUCTAL SECRETORY PROTEINS AS A MEDIUM SUPPLEMENT FOR IN VITRO EMBRYO PRODUCTION IN CATTLE

The purpose of this study was to determine the effect of heparin-binding oviducal secretory proteins as a media supplement on IVF and embryo development in cattle. The native proteins were isolated from oviducts collected from a slaughterhouse by repeated freeze-thawing and precipitated by ammonium sulfate (60%) followed by dialysis overnight in 10 mM phosphate buffer solution containing 1 mM PMSF at pH 7.0 at 4°C. The dialyzed product was then passed through a high-performance liquid chromatography system with a HiTrap™ heparin prepacked column (GE Healthcare, Piscataway, NJ) equilibrated with 10 mM phosphate buffer containing 1 mM EDTA. The collected protein fractions [heparin-binding protein (HBP), heparin-unbinding protein (HUBP)] and total protein (TP) were run on SDS-PAGE. The SDS-PAGE profile of the eluted HBP fraction contained 5 major protein bands visible between approximately 66 to 97 kDa, the profile of the HUBP fraction contained 2 bands nearer to approximately 66 kDa and in TP, all 7 bands were visible between approximately 60 to 95 kDa. All 3 fractions (i.e. HBP, HUBP and TP) were used in 3 different concentrations (0, 1, 10 and 30 μg mL–1) for in vitro maturation, sperm preparation, IVF and in vitro culture of cleaved embryos. Cumulus–oocyte complexes were collected from slaughterhouse ovaries, washed 4 to 5 times and cultured in maturation media for 24 h in a 5% CO2 incubator at 38.5°C with maximum humidity. In vitro-matured oocytes were co-incubated with capacitated sperm in Fert-BO media at 38.5°C in 5% CO2 in air with maximum humidity. After 18 h, oocytes were washed thoroughly and cultured in embryo development media for cleavage. After 40 to 42 h, cleavage was observed and embryos were transferred into the replacement media for further development. In the HBP group, overall cleavage rates (%) were 63 ± 3.0, 53 ± 2.6, 65 ± 4.7 and 43 ± 0.6 and blastocyst formation (%) was 17 ± 1.6, 23 ± 2.5, 25 ± 2.2 and 24 ± 1.7 in 0, 1, 10 and 30 μg mL–1 concentrations, respectively. However, in the HUBP group, the overall cleavage rates (%) were 66 ± 4.3, 56 ± 2.0, 60 ± 2.7 and 32 ± 0.8 and blastocyst formation (%) was 18 ± 1.1, 25 ± 1.8, 24 ± 2.2 and 14 ± 0.8 in 0, 1, 10 and 30 μg mL–1 concentrations, respectively. In the TP group, cleavage rates (%) were 67 ± 4.9, 60 ± 4.1, 73 ± 3.2 and 41 ± 1.6 and blastocyst formation (%) was 19 ± 1.1, 18 ± 1.9, 27 ± 2.2 and 20 ± 1.6 in 0, 1, 10 and 30 μg mL–1 concentrations, respectively. These results indicate that 10 μg mL–1 of the total oviductal secretory protein fraction used as a media supplement significantly increased (P < 0.01) the cleavage rate and blastocyst formation as compared with the HBP and HUBP fractions. The authors sincerely acknowledge the director and joint director (R), National Dairy Research Institute, Karnal and Incharge, Eastern Regional Station, National Dairy Research Institute, Kalyani, India, for providing the facilities to carry out the work.

Coenzyme Q10 content in follicular fluid and its relationship with oocyte fertilization and embryo grading

No data are available on the presence and content of Coenzyme Q10 (CoQ10) in human follicular fluid and its role. To assess the presence and concentration of CoQ10 in human follicular fluid in relation to oocyte fertilization. CQ10 content was measured in follicular fluid obtained from 20 infertile women undergoing ovarian stimulation program for in vitro fertilization. CoQ10 levels were assayed by high-performance liquid chromatography system and normalized for follicular cholesterol and protein levels. Oocyte morphology and embryo grading were assessed. CoQ10/Protein levels resulted significantly in mature versus dysmorphic oocytes. Similarly, CoQ10/Cholesterol was significantly higher in grading I-II versus grading III-IV embryos. This study is the first demonstration of the presence of CoQ10 in the human follicular fluid. Although the biological and endocrine mechanism of CoQ10 in the follicular fluid and its correlation with oocyte and embryo development is unclear, a new step may be the administration of CoQ10 in infertile women to evaluate the biological and reproductive outcomes.

Determination of alendronate in low volumes of plasma by column-switching high-performance liquid chromatography method and its application to pharmacokinetic studies in human plasma

A sensitive and simple column-switching high-performance liquid chromatographic (HPLC) method with fluorescence detection was developed for the determination of alendronate in human plasma. Alendronate and pamidronate (internal standard, IS) in plasma including Na(2)EDTA were precipitated with sodium hydroxide and calcium chloride after deproteinization using 10% trichloroacetic acid solution. The precipitated alendronate and IS were reconstituted by sodium citrate and citric acid and then derivatized with 9-fluorenylmethyl chloroformate. The resulting solution was injected onto an HPLC system consisting of a pretreatment column and an analytical column, which were connected with a six-port switching valve. The assay was linear in the concentration range of 2-100 ng/mL in 1 mL of plasma with high precision and accuracy, and the limit of detection was 0.5 ng/mL. It was successfully applied to evaluate the pharmacokinetic parameters of alendronate in human volunteers following single oral administration. The mean value of maximum alendronate plasma concentration (C(max)) was 37.69 ng/mL, and the mean time to reach the C(max) (T(max)) was 1.08 h. The area under the plasma concentration-time curve (AUC) and elimination half-life (T(1/2)) were 106.48 ng/mL/h and 1.66 h, respectively.

Comprehensive two-dimensional high performance liquid chromatography system with immobilized liposome chromatography column and monolithic column for separation of the traditional Chinese medicine Schisandra chinensis

A comprehensive two-dimensional (2D) separation is one that employs two separation dimensions (columns) and draws on all of the available resolving power from each of the dimensions of separate the components in a sample. In this study, a comprehensive 2D chromatography approach was developed for the separation and identification of membrane permeable compounds in a famous traditional Chinese medicine of Schisandra chinensis. The first dimensional column was the immobilized liposome chromatography (ILC) column, which mimics the biological membranes and can be used to study drug-membrane interactions in liquid chromatography. Using an automatic ten-port switching valve equipped with two sample loops, the section of the first-dimension was introduced in the second-dimension consist of a silica monolithic column. More than 40 components in Schisandra chinensis were resolved by using the developed separation system and among them 14 compounds were identified interacting with the ILC column based on their retention action, UV and mass data. With this comprehensive 2D-HPLC system, the three-dimensional chromatographic fingerprints of Schisandra chinensis were preliminarily established and processed by using principal component analysis and hierarchical clustering analysis. The obtained information can distinguish the unacceptable samples of the quality control. The result demonstrated that the 2D biochromatography system has been demonstrated to have more advantages of finding strong binding bioactive components, providing an enhanced peak capacity, good sensitivity and powerful resolution biological fingerprinting analysis of complex TCMs, which was a useful means to control the quality of and to clarify the membrane permeability of the compounds in Schisandra chinensis.

Speciation of trace elements in human serum by micro anion exchange chromatography coupled with inductively coupled plasma mass spectrometry

Speciation analysis of essential trace elements in human serum provides important information on nutritional status and homeostatic mechanisms regulating transport processes, acute phase reactions, and protection against oxidative damage. Anion exchange high-performance liquid chromatography (HPLC) combined with inductively coupled plasma mass spectrometry (ICP–MS) has proved to be a useful tool in speciation. Here we describe a fast method that can be applied to carry out the speciation of Fe, Cu, Zn, and Se in as little as 1 ml of serum. The method employs monolithic anion exchange micro columns installed on a tandem HPLC system coupled on-line with an ICP–MS detector. The chromatographic separation is similar to those reported previously but with considerable gain in terms of time and sample requirement. Reproducibility is acceptable for most species. Using our method, we were able to find species-specific differences between different commercially available trace element reference materials. Because the method chosen to collect blood might interfere with speciation, the proposed methodology was used to compare heparinized plasma, ethylenediaminetetraacetic acid (EDTA) plasma, and serum from adult healthy volunteers. As expected, EDTA strongly affects speciation analysis (especially for Fe and Zn), whereas changes due to the use of lithium–heparin (Li–He) as anticoagulant appear to be minimized.

Implementation of a cost-effective HPLC/UV-approach for medical routine quantification of donepezil in human serum

A novel, simple, specific and sensitive high performance liquid chromatography (HPLC) assay for the detection and quantification of donepezil in serum of demented patients has been developed and validated. The analytical procedure involves an offline serum preextraction using solid phase extraction (SPE) cartridges (Oasis ® HLB, Waters Co). The chromatographic analyses were performed on a Dionex HPLC system with a Phenomenex Luna Phenyl-Hexyl analytical column, and a mobile phase with the two components 0.02 mol/l phosphate buffer and acetonitrile. The flow rate was 0.4 ml/min. For the detection of donepezil three different UV wavelengths were used as an interference-control check. Interference tests between donepezil and 100 of the most commonly used concomitant medications allow quantification of donepezil under the polypharmaceutical conditions of the daily clinical routine. The retention time for donepezil was 12.1 min. The method was validated according to the guidelines of the Society of Toxicology and Forensic Chemistry (GTFCh): The calibration curve was linear over a concentration range from 5 to 160 ng/ml ( n = 8/ r 2 > 0.999). No endogenous compounds were found to interfere with the analyte, which was shown by retention times for the comedication most often prescribed to demented patients. The method had an accuracy of >85%. Intra- and inter-assay coefficients of variation were <6% and <8%, respectively, at three different concentrations. The limit of quantification (LOQ) and the limit of detection (LOD) were found to be 6.1 and 1.7 ng/ml for donepezil. Application of the method to patient serum samples discovered that concentrations suggested as “therapeutic” in the literature may only be reached either by high, off-label dosages or by utilization of inhibitory metabolic effects of the comedication.

Effect of enhanced voluntary physical exercise on brain levels of monoamines in Huntington disease mice

Using the R6/1 mouse model of Huntington disease (HD), we have recently shown that voluntary physical activity was able to correct the depressive-like behaviours exhibited by the HD animals at a pre-motor symptomatic stage of the disease. Using the high performance liquid chromatography system, we have now evaluated the effect of exercise on monoamine metabolism in HD mice. We found that serotonin and its metabolite as well as dopamine and noradrenaline were reduced across several brain regions in female R6/1 animals. Our data also suggest that some of these neurochemical deficits were modulated by physical activity, in a genotype-region dependent manner. These newly identified changes could account for some of the behavioural effects of exercise previously reported in HD mice.

Pharmacological Classification of Drugs by Principal Component Analysis Applying Molecular Modeling Descriptors and HPLC Retention Data

Pharmacological classification of drugs by principal component analysis (PCA) based on molecular modeling and high-performance liquid chromatography (HPLC) retention data is proposed. First, a group of 20 drugs of recognized pharmacological classification are chromatographed in eight diversified HPLC systems, applying columns with octadecylsilanes, phosphatidylcholine, as well as α1-glycoprotein and albumin. Additionally, molecular modeling studies, based on the structural formula of the drugs considered, are performed. Sixteen structural descriptors are derived. A matrix of 20 × 24 HPLC data together with molecular parameters are subjected to principal component analysis, and this revealed five main factors with eigenvalues higher than 1. The first principal component (factor 1) accounted for 47.8% of the variance in the data, and the second principal component (factor 2) explained 21.0% of data variance. The total data variance was 82.6% and is explained by the first three factors. The clustering of drugs is in accordance with their pharmacological classification, which proved that the PCA of the HPLC retention data, together with their structural descriptors, allowed the drugs to be segregated accurately to their pharmacological properties. This may be of help in reducing the number of biological assays needed in the development of a new drug.

Ultra high‐performance liquid chromatography of porphyrins in clinical materials: column and mobile phase selection and optimisation

Ultra high-performance liquid chromatographic (UHPLC) systems on columns packed with materials ranging from 1.9 to 2.7 µm average particle size were assessed for the fast and sensitive analysis of porphyrins in clinical materials. The fastest separation was achieved on an Agilent Poroshell C(18) column (2.7 µm particle size, 50 × 4.6 mm i.d.), followed by a Thermo Hypersil Gold C(18) column (1.9 µm particle size, 50 × 2.1 mm i.d.) and the Thermo Hypersil BDS C(18) column (2.4 µm particle size, 100 × 2.1 mm i.d.). All columns required a mobile phase containing 1 m ammonium acetate buffer, pH 5.16, with a mixture of acetonitrile and methanol as the organic modifiers for optimum resolution of the type I and III isomers, particularly for uroporphyrin I and III isomers. All UHPLC columns were suitable and superior to conventional HPLC columns packed with 5 µm average particle size materials for clinical sample analysis.

Chemical composition changes of post-harvest coconut inflorescence sap during natural fermentation

Coconut inflorescence sap (CIS) is sweet, oyster-white and translucent and was reported to be highly nutritive and a good digestive agent. The chemical composition changes including total sugar, reducing sugar, ethanol, total acidity, volatile acid, amino acid, vitamin C and total phenolic contents of postharvest coconut inflorescence sap (PCIS) were investigated during a 12-day natural fermentation, and the variety and content of phenolic compounds of fresh coconut inflorescence sap (FCIS) and natural fermented coconut inflorescence sap (NCIS) were also studied by an high performance liquid chromatography (HPLC) system. Total acid, volatile acid and total phenolic contents increased during natural fermentation, while total sugar contents decreased during natural fermentation. The amino acid content declined steadily after harvesting until 3 days, but then remained almost constant. Vitamin C content decreased on day 1, slowly rose to 20.7 mg/L on day 3, and then decreased obviously. Five kinds of phenolic compounds were detected by HPLC. These compounds all increased in NCIS compared with FCIS; both NCIS and FCIS also contained other kinds of phenolic compounds.

Comparison and evaluation of steroid alkaloid solasodine on in vivo and in vitro cultures of Solanum surattense Burm L.

Steroid alkaloid solasodine is a nitrogen analogue of diosgenin that is used as a raw material for synthesis of steroid drugs. Plant medicinal Solanum surattense (Solanaceae) is a rich source of solasodine. The purpose of this study was production and maintenance of callus from leaves and comparison of solasodine steroid alkaloid production in outdoor plants and in vitro calli. The leaves were collected, sterilized and placed on MS medium supplemented with BAP (1 to 2 mg/L) and NAA (1 to 3 mg/L). The samples were kept in a growth chamber at 25°C with 16/8 (day/night) photoperiod and irradiance of 3000 to 2000 lux. The methanol extracts was performed to obtain solasodine from 2 g dry weight of each leaves and calluses and then injected into the high-performance liquid chromatography (HPLC) system. The results showed that solasodine produced by leaves was 0.3316 mg/g whereas, the solasodine content obtained from calluses were 0.3951 mg/g (in 1 mg/L BAP + 2 mg/L NAA) and 0.3908 mg/g (in 1 mg/L BAP + 3 mg/L NAA). Plant tissue cultures have been suggested as a potential tool for solasodine production. Key words: Callus induction, Solanum surattense Burm, in vitro, in vivo, solasodine,high-performance liquid chromatography (HPLC).

English

A new, simple, specific, accurate and precise reversed phase-high performance liquid chromatography (RP-HPLC) method was developed for the determination of levofloxacin in rat plasma and saliva was developed. An HPLC system based on a Phenomenex Luna C18Column (250 × 4.6 mm) and a UV detector (λ = 296 nm) were used. A mixture of Acetonitrile: water (80:20 v/v) adjusted to pH 3.5 by orthophosphoric acid at a flow rate of 1.4 ml/min was used as mobile phase. The proteins were precipitated with methanol. The average recovery was 94.79 and 92.66%, respectively in plasma and saliva. The detection limit for levofloxacin in plasma and saliva was 1 µg/ml. The calibration curve was linear over the concentration range 1 to 16 mg/ml for plasma and saliva. The inter-day and intra-day assay coefficients of variation were found to be less than 5%. The present validated method was successfully used for pharmacokinetic studies of levofloxacin in plasma and saliva. Key words: High-performance liquid chromatography (HPLC), levofloxacin, plasma, saliva, pharmacokinetics.

A miniature mass spectrometer for liquid chromatography applications

A miniature mass spectrometer capable of detecting analytes eluting from a high-performance liquid chromatography (HPLC) system is described and demonstrated for the first time. The entire instrument, including all pumps and the computer, is contained within a single enclosure that may be conveniently accommodated at the base of the HPLC stack. The microspray ion source, vacuum interface, ion guide, and quadrupole ion filter are all microengineered. These components are fabricated in batches using microelectromechanical systems (MEMS) techniques and considered to be consumables. When coupled to a standard HPLC system using an integrated passive split, the limit of detection for reserpine while scanning the full mass range is 5 ng on-column (1 pg of which is passed to the microspray). The mass range is m/z 100-800, and each spectrum is typically acquired at a rate of 1 scan per second.

Effects of coenzyme Q10 and ^|^alpha;-lipoic acid supplementation in fructose fed rats

This study was conducted to investigate the effects of α-lipoic acid and coenzyme Q10 on plasma levels of lipids, asymmetric dimethylarginine, oxidative stress in fructose fed rats which provide a model of dietary-induced insulin resistance and to evaluate vascular changes developing in these rats by histologically. Male Sprague Dawley rats were used in this study. The animals were divided into 4 groups. Group 1 did not receive any medication and served as a control. Group 2 received a regular diet and water ad libitum and fructose was administered as % 10 solution in drinking water. Group 3 received α-lipoic acid (100 mg/kg/day) i.p. for 5 weeks and Group 4 received coenzyme Q10 (10 mg/kg/day) i.p. for 5 weeks. For determination of plasma asymmetric dimethylarginine, glutathione and malondialdehyde levels, high-performance liquid chromatography system was used. Homeostatic model assessment as a measure of insulin resistance was calculated. Lipid profile measurements were determined using enzymatic assay on an Auto analyzer. The high fructose diet was significantly associated with an increase in levels of plasma LDL, VLDL and total cholesterol and decrease in level of HDL cholesterol. Plasma asymmetric dimethylarginine, malondialdehyde and glutathione levels were also increase in these rats. α-lipoic acid or coenzyme Q10 supplementation was found to have some positive effect on these parameters. These findings were also demonstrated by morphological observation of the aorta. We demonstrated that administration of α-lipoic acid and coenzyme Q10 notably suppresses oxidative and nitrative stress, hyperinsulinemia, insulin resistance developing in fructose fed rats, a model of metabolic syndrome (MS). These positive effects of α-lipoic acid or coenzyme Q10 can be attributed to its antioxidant activity.

Comparison and evaluation of a novel bioassay and high‐performance liquid chromatography for the clinical measurement of serum voriconazole concentrations

The aim of this study was to develop and validate a novel bioassay for determining serum voriconazole (VRC) concentrations and to compare its routine clinical performance with that of high-performance liquid chromatography (HPLC). The biological activity of VRC was measured by a plate diffusion assay using a VRC-hypersusceptible Candida kefyr strain. The bioassay's utility was tested by measuring steady-state VRC concentrations in 100 serum probes from VRC-treated patients. The HPLC system used solvent extraction with hexane:dichloromethane followed by reversed-phase HPLC with ultraviolet detection. The intra-day and inter-day accuracy of the bioassay was <5%, while that of HPLC was <1%. The precision (mean coefficient of variation, 3.5%) was equal for both the methods. The limit of quantification was lower for HPLC (0.2 mg l(-1)) than for the bioassay (0.5 mg l(-1)). The result of linear regression analysis was HPLC = 1.0178 (bioassay) + 0.328; R(2) = 0.88; n = 100. Results of the serum panel ranged from 0.5 to more than 8.0 mg l(-1) for the bioassay and from 0.26 to 10.1 mg l(-1) for HPLC. Especially in laboratories without access to HPLC, the bioassay may be a clinically useful tool for therapeutic drug monitoring.

Uncovering the Proteome Response of the Master Circadian Clock to Light Using an AutoProteome System

In mammals, the suprachiasmatic nucleus (SCN) is the central circadian pacemaker that governs rhythmic fluctuations in behavior and physiology in a 24-hr cycle and synchronizes them to the external environment by daily resetting in response to light. The bilateral SCN is comprised of a mere ~20,000 neurons serving as cellular oscillators, a fact that has, until now, hindered the systematic study of the SCN on a global proteome level. Here we developed a fully automated and integrated proteomics platform, termed AutoProteome system, for an in-depth analysis of the light-responsive proteome of the murine SCN. All requisite steps for a large-scale proteomic study, including preconcentration, buffer exchanging, reduction, alkylation, digestion and online two-dimensional liquid chromatography-tandem MS analysis, are performed automatically on a standard liquid chromatography-MS system. As low as 2 ng of model protein bovine serum albumin and up to 20 μg and 200 μg of SCN proteins can be readily processed and analyzed by this system. From the SCN tissue of a single mouse, we were able to confidently identify 2131 proteins, of which 387 were light-regulated based on a spectral counts quantification approach. Bioinformatics analysis of the light-inducible proteins reveals their diverse distribution in different canonical pathways and their heavy connection in 19 protein interaction networks. The AutoProteome system identified vasopressin-neurophysin 2-copeptin and casein kinase 1 delta, both of which had been previously implicated in clock timing processes, as light-inducible proteins in the SCN. Ras-specific guanine nucleotide-releasing factor 1, ubiquitin protein ligase E3A, and X-linked ubiquitin specific protease 9, none of which had previously been implicated in SCN clock timing processes, were also identified in this study as light-inducible proteins. The AutoProteome system opens a new avenue to systematically explore the proteome-wide events that occur in the SCN, either in response to light or other stimuli, or as a consequence of its intrinsic pacemaker capacity.