PROPERTIES AT A GLANCE

• Zivak- LC-MS/MS amino acid analysis kit is designed and manufactured for the detection and quantitation of free amino acids in body fluids.
• Zivak- LC-MS/MS amino acid analysis kit enables the quantitation of 42 molecules including amino acids and metabolic intermediates in a single run.
• Amino acids can be detected and quantitated without a deproteinization step in body fluids with a complex matrix
• The amino acid determinations have a very high analytical sensitivity (LOD; 0.007-0.60 nmol/mL, LOQ; 0.02-1.8 nmol/mL).
• The amino acid determinations have a very high specificity of 100 %.
• The method is very suitable for clinical laboratory settings due to a short sample preparation time of 10 minutes and a run time of 18 minutes. 3 samples within one hour and 25 samples within a day (8 hours) can be analyzed.
• It is a powerful screening test which 42 analytes can be analyzed with a single run.
• It is a reliable confirmation test due to its high analytical accuracy, precision and very low limit of detection
• Zivak- LC-MS/MS amino acid analysis kit can be used in a wide variety of cases resulting in primary and secondary aminoacidurias, such as;
  • Screening of inborn errors of metabolism in newborns,
  • In the diagnosis of renal tubular transport system defects, dieases of liver and metabolism of children and adults,
  • Confirmation of screeninig tests. 
• Zivak- LC-MS/MS amino acid analysis kit is produced in accordance with the TS ISO 9001:2000 TS ISO 13485:2003 quality standards.
  
  

INTENDED USE

The detection and quantitation of amino acids has a key role in the screening, diagnosis and follow-up of patiens with various pathologies which result in primary or secondary aminoacidurias, such as inborn errors of metabolism and dieaeses effecting renal tubular transport system or the liver.

Historically, colorimetric tests, Guthrie and some chromatographic tests have been used in the determination of amino acids. Nowadays, tandem mass spectrometry (MS/MS) has replaced these methodologies due to its higher specificity and analytical sensitivity.

This method is suitable for screening purposes due to capability of analyzing a multitude of analytes within a single run, and is suitable for use as a confirmatory method due to its high specificity and very low limits of detection for these analytes.

SAMPLE AND CALIBRATOR PREPARATION PROCEDURE

Preparation of calibrators

Calibrators are prepared daily and run through sample preparation procedures with the patient samples.

LEVEL I: 25 μl STD1 + 25 μl STD2 + 50 mL distilled water

LEVEL II       : 50 μl STD1 + 50 μl STD2

Working procedure

1. Add 100 mL each of patient samples, Level I and II calibrators to glass sample preparation tubes

2. Add 100 mL of Reagent 1 and vortex for 10 seconds

3. Add 200 mL of Reagent 2 and vortex for 10 seconds

4. Add 200 mL of Reagent 3  and vortex for 10 seconds.  Wait for 2 minutes and vortex for 10 seconds. After mixing, wait for 1 more minute.  

5. Add 200 mL of Reagent 4  and and vortex for 10 seconds. After the mixing, wait for 1 minute.  

6. Centrifuge for 3 minutes at 4000 rpm.

7. Pipet 200 μL from the supernatant, to a vial and evaporate under Nitrogen gas flow. 

8. Add 500 μL of Reagent5 and vortex for 10 seconds.

At this step, the samples maintain their stability for 24 h at room temperature, 3 days at 2-4 °C.

 9. Transfer the samples to the loader and inject 10 mL of sample or

Calibrator to the Tandem GoldLC-MS/MS.

TABLE 1: Amino acid measurement parameters

Amino Acid	Symbol	MW	RT	MH+ (m/z)	MS/MS	CE (eV)	LOD (nmol/mL)
Arginine	ARG	174	3,36	303	156	15V	0,138
Homoarginine (IS)	HARG	225	3,52	317	84	30V	-
Glutamine	GLN	146	3,92	275	172	15V	0,099
Citrulline	CIT	175	3,99	304	156	15V	0,087
Anserine	ANS	240	4,14	369	309	20V	0,15
Hydroxylysine	HYL	198	4,30	317	171	10V	0,393
Serine	SER	105	4,38	234	104	15V	0,438
Asparagine	ASN	132	4,46	260	157	15V	0,153
3-methyl-L-histidine	3-MeHIS	169	4,64	298	256	20V	0,072
Hydroxyproline	HYP	131	4,69	260	172	10V	0,063
N-methyl-L-histidine	N-MeHIS	169	4,74	298	210	20V	0,039
Glycine	GLY	75	4,96	204	76	8V	0,174
Threonine	THR	119	5,04	248	74	15V	0,132
Beta alanine	SAR	89	5,97	218	116	8V	0,051
Alanine	ALA	89	6,05	218	130	10V	0,192
Gamma-aminobutyric acid	GABA	103	6,57	232	172	10V	0,252
Sarcosine	BALA	89	6,87	218	116	8V	0,09
Beta-aminoisobutyric acid	BAIB	103	7,12	232	172	10V	0,12
Alpha-aminobutyric acid	ABA	103	7,54	232	144	15V	0,063
Ornithine	ORN	168	7,80	347	156	15V	0,036
Carnosine	CAR	226	7,96	441	284	20V	0,045
Methionine D3 (IS)	MET-D3	152	8,20	281	193	10V	-
Methionine	MET	149	8,28	278	142	15V	0,081
Proline	PRO	115	8,43	244	156	15V	0,15
Lysine	LYS	182	9,05	361	170	15V	0,054
Aspartic Acid	ASP	133	9,06	304	216	15V	0,204
Histidine	HIS	155	9,20	370	196	15V	0,144
Thiaproline	THPR	133	9,28	262	174	10V	0,024
Valine	VAL	117	9,49	246	158	15V	0,315
Glutamic Acid	GLU	147	9,67	318	172	15V	0,54
Tryptophan	TRP	204	10,19	333	245	15V	0,006
Alpha-aminoadipic Acid	AAA	161	10,92	332	98	15V	0,159
Leucine	LEU	131	11,35	260	172	10V	0,138
Phenylalanine	PHE	165	11,52	294	164	15V	0,144
Isoleucine	ILEU	131	11,85	260	130	10V	0,102
Norleucine	NLEU	131	12,25	260	172	10V	0,186
Cysteine	CYS	121	12,38	336	162	15V	0,048
Adrenaline	ADR	183	12,53	424	252	15V	0,081
Alpha-aminopimelic Acid	APA	175	12,67	346	198	15V	0,084
Cystathionine	CTH	222	12,96	479	230	15V	0,012
Homophenylalanine (IS)	HPHE	216	13,38	308	117	20V	-
Cystine	C-C	240	13,47	497	248	15V	0,021
Homocysteine	HCYS	135	13,62	350	142	15V	0,072
Tyrosine	TYR	181	14,36	396	222	15V	0,063
Homocystine	HC-CH	268	15,90	525	262	15V	0,009

CHROMATOGRAMS

Product Catalog no: ZV-…………	Amino acid LC-MS/MS Analysis Kit
	Blood-Serum-Plasma and other body fluids (200 tests)
Contents
Reagents
Catalog No:	Product	Volume	Quantity
ZV-3002-0200-10ST1	Standard Reagent 1 (STD 1)	2 mL	2 /kit
ZV-3002-0200-10ST2	Standard Reagent 2 (STD 2)	2 mL	2 /kit
ZV-3002-0200-10R1	Reagent1 (R1, Int Std)	20 mL	1 /kit
ZV-3002-0200-10R2	Reagent2 (R2)	40 mL	1 /kit
ZV-3002-0200-10R3	Reagent3 (R3)	40 mL	1 /kit
ZV-3002-0200-10R4	Reagent4 (R4)	40 mL	1 /kit
ZV-3002-0200-10R5	Reagent5 (R5)	50 mL	2 /kit
ZV-3002-MA	Mobile Phase A	1 L	1 /kit
ZV-3002-MB	Mobile Phase B	1 L	1 /kit
Materials
ZV-9017-0200	2 mL glass sample preparation tubes	200 /pack	1 /kit
ZV-9001-0100	Vials (1,8 mL)	100/ pack	2 /kit
ZV-9002-0100	Vial Lids (1,8 mL)	100/ pack	2 /kit
ZV-9010-0003	Sample holder		1 /kit
ZV-9003-1000	20-200 µL  pipet tips	1000/pack	2 /kit
ZV-9004-0250	100-1000 µL  pipet tips	250/pack	1/kit
ZV-9014-0001	20-200 µL pipet		1 /kit
ZV-9015-0001	100-1000 µL pipet		1 /kit
ZV-3002-C	Zivak-Amino Acid column		1 /kit
ZV-3002-BR-Rev.1	User Manual		1  /kit
Materials not supplied
	Vortex
	Tandem GoldLC-MS/MS instrument
	Centrifuge (for eppendorf tubes) Nitrogen gas
Storage and Stability
	Standard  1 and 2 reagents should be kept at -20 °C. R1, R2, R3 and R4 reagents should be keptat 4 °C. All other reagents can be kept at room temperature. When kept as suggested in the user manual, all reagents will be stable until the expiry date.
Warnings and precautions
	As a general precaution all sample preparation steps should be carried out in a safety hood, wearing protective gloves and glasses. When working with biological samples, necessary steps should be taken to avoid contamination with bloodborne pathogens. All samples posing a biological threat should be appropriately disposed of.