GC MS LC MS/MS SELDI-TOF CEMS HPTLC MS ICPMS TGAMS

Niemann-Pick C1 Disease from Human Plasma

Adrenal Cortical Dysfunction by Liquid Chromatography-Tandem Mass Spectrometry

Cancer using SELDI-TOF MS Propionic Acedemia using GCMS Diagnosing Diabetes Diagnosis of vitamin B12 deficiency Diagnosis of neuroblastomas etc.
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PKU is a metabolic disorder caused by a deficiency of the liver enzyme phenylalanine hydroxylase Phenyalanine to Tyrosine

Fig. 1. A drop of blood is spotted onto a sample card, and an aliquot is extracted for analysis by MS or GC-MS. The peak profile is used to diagnose metabolic errors.
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Amino acids extraction 200 L methanol/HCl 0.1% in a screw cap vial, 1 h, at 4C or by sonication for 1 min. 100 L of the extract placed in a vial, Addition of the internal standard and removing of the reagent ina nitrogen stream

AA derivatization
Esterification: 500 Lbutanol: acetyl chloride, 4:1 (v/v), 30 min, at 100C. The excess reagent is removed with a stream of nitrogen
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Analytical instrument : Trace DSQ ThermoFinnigan quadruple mass spectrometer Trace GC. Column capillary column Rtx-5ms 30 m 0.25 mm, 0.25 m TC Prog. : : 50 C (1 min) to 310 C, at 20 C/min. Carrier gas : Helium (99.9995%) Flow rate : 1 mL/min

MS Ion source : EI mode SIM mode Scan m/z 50 to 500

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Fig. 2. The mass spectra of the phenylalanine and tyrosine as trifluoroacetic butyl ester derivatives used for PKU diagnosis
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The fingerprint chromatograms in SIM-GC-MS for a PKU patient (our results) present the differences between the first (top chromatogram) and the third month of treatment (second chromatogram) . The retention times for the amino acids: 15N-Gly at 6.40 min, Val at 7.02 min, Leu at 7.50 min, Pro at 8.40 min, Phe at 9.70 min and Tyr at 11.28 min. 10

PKU diagnosis could be tested by calculating the ratio Phe/Tyr using 20 L of blood spots. A comparison of two chromatograms demonstrates the benefit of the treatment in the case of a PKU child . The first fingerprint chromatogram presents higher intensity of the phenylalanine peak in the case of a patient presenting PKU, and after three months of treatment the peak of Phe is significantly decreased. The ratio of Phe/Tyr decreased from 2.30 at 1.02.

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What is Niemann-Pick Disease? - Neurodegenerative disease - metabolic dysfunction

Why LC-MS/MS?

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How is it Diagnosed? Monitoring oxidation products of cholesterol in plasma

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Fig.1 Spectra for cholestane-5-hydroxy3,6-bis (dimethylglycinate) (CH-DMG2)

Fig.2 Spectra for 7-ketocholesterol dimethylglycinate (KC-DMG)

Analysis:
- Sample preparation Derivatization by N,N-Dimethyl glycine -LC-MS/MS APCI MRM scan Mode

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What is SELDI-TOF MS ?

Difference between the mass spectrometric proteomic patterns of serum

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Take 1-3 l serum sample Spot sample on the activated protein chip array

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Also known as Kimmelstiel-Wilson syndrome

Renal disease

Caused by angiopathy of capillaries in the kidney glomeruli

Risk factors- elevated urinary albumin excretion, increased blood pressure, and poor glycemic control

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To extract maximal information proteins present in small quantities

CE-ESP-MS: Fast, sensitive, automated

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Detection of the current status of diabetic nephropathy of diabetic patients. Differentiation between patients with diabetes mellitus and healthy controls in general. Evaluation of CE-MS-defined patterns derived from urinary polypeptides of patients with diabetic nephropathy differ from those of patients with other chronic renal diseases.

Making a statement of the progression of diabetic nephropathy.

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Validation of the treatment success of drugs, like candesartan in context of diabetic nephropathy

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Analogue insulin.

Animal insulin :-bovine insulin and porcine insulin.

Human insulin

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Planar chromatography is well suited for peptide analysis. Analysis of intact proteins. Simple to medium complex mixtures. Peptides from tryptic digest can be separated by HPTLC and analyzed by MS.

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Human insulin

Bovine insulin

Porcine insulin This were all diluted in 0.1% TFA (1mg/ml)

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HPTLC silica gel Si 60 F254 20 10 cm. Automated TLC Sampler 4 Band length :- 5 mm Track distance :-10 mm Application volume :- 5 micro litre.

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Human insulin 968,7816[M + 6H]6+ 1162,5367[M+5H]

Porcine insulin 963,78[M+6H]6+ 1156,3347[M+5H]

Bovine insulin 956,4421 [M+6H] 1147,5288[M+H5H]

1452,6680[M+4H]
1936,5513[M+3H]

1445,1658[M+4H]
1926,5491[M+3H]

1434,1584[M+4H]
1912,2065[M+3H]

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Provide rapid, comprehensive, multi-component analyses Maintain or improve sensitivity, selectivity, and accuracy Replacing many older traditional techniques

Is becoming an indispensable tool in clinical laboratory

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THANK YOU

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