Protein Precipitation to Remove Carbohydrates that Interfere in Protein-Bound Tryptophan Quantification in Soybean Seeds

Tryptophan is one of the nine essential amino acids in humans that can only be obtained through diets and supplements.It is a precursor to many biological processes,such as serotonin,melatonin,kynurenin,and niacin(nicotinamide)vitamin synthesis.The content of tryptophan in foods,such as soybean is an important indicator of nutritional value.Therefore,accurate quantification of tryptophan in soybean is crucial to soybean nutritional improvement.Quantification of soybean protein-bound amino acids first involves acid hydrolysis of total protein to liberate amino acids.However,tryptophan quantification following acid hydrolysis is difficult or impossible due to its reactions with soybean carbohydrates.Therefore,removal of carbohydrates from soy proteins prior to acid hydrolysis is necessary.In this study,we compared four common protein precipitation methods(i.e.,methanol,acetonitrile,acetone,and trichloroacetic acid(TCA)protein precipitation methods)to determine the best method to separate soy proteins from carbohydrates,and concluded that acetone provided the highest recovery of soy proteins.Tryptophan content in the precipitated proteins was determined after acid hydrolysis of the proteins using liquid chromatography-tandem mass spectrometry multiple reaction monitoring(LC-MS/MS-MRM).No significant difference in the tryptophan content was found among proteins precipitated with methanol,acetonitrile,and TCA,suggesting that these precipitated proteins have similar compositions.A slightly lower,but statistically significant tryptophan content was found in the acetonitrile-precipitated proteins,suggesting that these proteins contain slightly higher glycosylated proteins.

Extraction of Keratin Protein from Chicken Feather

The present research was conducted to extract keratin protein from chicken feathers. Protein is an important nutrient needed by our body to maintain body structures and is an important ingredient for cosmetic products. Chicken feathers have high level of keratin protein content and can become a suitable protein source. The main processes involved are first dissolving chicken feathers using different reducing agents and later on separating the protein from chemicals. Reducing agents used are potassium cyanide, thioglycolic acid and sodium sulphide. Once the feathers are dissolved using reducing agents, ammonium sulfate solution is added to the solution for the precipitation of protein. The precipitated protein is washed with water several times and sodium hydroxide solution is used to obtain protein back in the solution form. Out of three different reducing agents used, sodium sulfide gives the highest efficiency in dissolving chicken feathers since the feathers are dissolved in a very short period of time. The percentage of keratin protein is evaluated by means of biuret test and FTIR analysis. The analysis by FTIR confirmed the presence of carboxyl acid and amino groups in the protein solution. The biuret test helps in determining the concentration of protein obtained from different methods. Thus these two tests confirm the presence of protein in the solution. From this research, it can be concluded that protein can be extracted from chicken feathers. The keratin protein solution can be used for several purposes such as anti-aging cream, shampoo, and conditioner and for medical purposes such as bone replacement and bone graft.

Rapid Determination of Ranitidine in Human Plasma by Liquid Chromatography-Tandem Mass Spectrometry and Its Application to a Clinical Pharmacokinetic Study

A rapid and sensitive assay based on high performance liquid chromatography-tandem mass spectrometry（LC-MS/MS） was developed for the determination of ranitidine（RAN） in human plasma with codeine as internal standard（IS）.After protein precipitation with acetonitrile,the analyte and IS were separated on a Zorbax SB-Aq C18 column（150 mm×4.6 mm i.d.,5 μm） eluted with a mobile phase consisting of methanol/acetonitrile/10 mmol/L ammonium acetate containing 1% formic acid（pH=2.4）（volume ratio 12.5：12.5：75） at a flow rate of 1.0 mL/min.Detection was performed by electrospray ionization in the positive ion mode followed by the multiple reaction monito-ring（MRM） of the transitions of RAN at m/z 315.1→176.3 and of IS at m/z 300.1→165.1.The method was linear over a concentration range of 1―1000 ng/mL（r=0.9991） with a lower limit of quantitation（LLOQ） of 1 ng/mL and a limit of detection（LOD） of 0.3 ng/mL.Accuracy as relative error was from-0.01% to-1.7% and intra-day and inter-day precisions as relative standard deviation were ≤8.9% and ≤5.5%,respectively.The method was successfully applied to a pharmacokinetic study of ranitidine,getting a single oral dose（160 mg） to healthy volunteers.

Optimization of Protein Extraction and Decoloration Conditions for Tea Residues

To optimize alkaline method for extracting proteins from tea residue(TR), the effect of extraction conditions on tea protein extraction rate(TPER) was investigated. Single factor experiment showed the extraction temperature 80 °C, extraction time 100 min, p H value 13 and liquid–solid ratio 40:1 as the optimal extraction conditions. The orthogonal test revealed that the maximum TPER reached 29.71% under the following optimal combination of conditions: extraction temperature 70 °C, extraction time 60 min, p H 12 and liquid–solid ratio 50:1. For optimizing the purification of tea residue proteins, isoelectric point precipitation(p I), ammonium sulfate precipitation(a S) and isoelectric point plus ammonium sulfate precipitation(i PAS) were compared. The result showed that the highest protein precipitation rate(PPR) was 89.70% which was generated by using i PAS. Furthermore, powdered activated carbon was chosen as the most suitable decolorant for the extracted proteins.

Development of a method for the quantitative determination of geniposide in rat plasma by liquid chromatography-tandem mass spectrometry with positive/negative ion-switching electrospray ionization and its application in a pharmacokinetic study in rats

Geniposide is a major bioactive constituent isolated from Gardeniajasminoides Ellis. To evaluate the pharmacokinetics of geniposide in pre-clinical studies, a rapid and specific liquid chromatography-tandem mass spectrometry （LC-MS/MS） method was developed and validated. After simple protein precipitation, geniposide was analyzed on a DiamonsilR C18 column with a mobile phase of 10 mM ammonium acetate and methanol （20：80, v/v） at a flow rate of 0.6 mL/min. Detection was performed in ＂Truncated＂ multiple-reaction monitoring （MRM） mode with positive electrospray ionization （ESI） at m/z 411→411 for geniposide, and MRM mode with negative ESI ionization at m/z 415→295 for puerarin （internal standard, IS）. Linearity was established in the concentration range from 10.0 to 5000 ng/mL. The extraction recoveries ranged from 84.8% to 90.5% at concentrations of 10.0, 500 and 4.5x 103 ng/mL. The lower limit of quantification （LLOQ） was 10.0 ng/mL with 50 ~tL plasma. The validated method was successfully applied to the pharmacokinetic study of geniposide in rats at a dose of 200 mg/kg by oral administration.

Rapid Determination of Tetrodotoxin in Human Plasma by Ultra Performance Liquid Chromatography-Tandem Mass Spectrometry

A sensitive analytical method was developed to determine tetrodotoxin（TTX） in human plasma samples using protein precipitation, followed by ultra performance liquid chromatography（UPLC） analysis coupled with tandem mass spectrometry（MS/MS） using 11-deoxytetrodotoxin（11-deoxyTTX） as an internal standard. The plasma samples were prepared using protein precipitation prior to being analyzed by UPLC-MSfMS to identify TTX over a zwitterionic-hydrophilic interaction liquid chromatography column. The retention time values of TTX and 11-deoxyTTX were 4.12 and 3.67 min, respectively. TTX and 11-deoxyTTX were monitored and quantitated on the basis of their ion transitions for their respective precursor ions to their product ions（i.e., m/z 320.0→162. l for TTX and m/z 304.0→176.0 for 11-deoxyTTX） in the multiple reaction-monitoring mode. The lower limit of quantification of this method was determined to be 0.0199 ug/mL. This method showed good linearity for plasma samples that contained TTX concentrations in the range of 0.0199--1.99 ng/mL. The specificity, precision, accuracy, matrix effect, and stability characteristics of this method were also examined. The intra-assay precision and accuracy ranged from 1.89% to 6.00% and from 92.21% to 100.00%, whereas the inter-assay precision and accuracy ranged from 0.64% to 7.75% and from 99.38% to 101.26%, respectively. This new method therefore represents a rapid, accurate, reliable, and highly sensitive method for the qualitative and quantitative analyses of a trace amount of TTX in human plasma samples.