CN1313818C - Method for detecting phosphate radical in urine - Google Patents

Abstract

The present invention uses ytterbium ions, catechol violet as a chromogen, and a HEPES buffer solution with a pH of about 7.0 to qualitatively and quantitatively directly measure the content of phosphoric acid in undiluted human urine. The determination process is simple, convenient, and easy to measure. The result is accurate. This method has high sensitivity and is not interfered by other components in urine, so it can be applied clinically.

Description

A method for detecting phosphate radicals in urine

Technical field:

The invention relates to a method for detecting phosphate radicals, in particular to a method for detecting phosphate radicals in urine.

Background technique:

Phosphate is involved in various important biomineralization processes, such as bone formation and pathological processes such as the origin of kidney stones. Therefore, the detection of phosphate in biological fluids is important. We know that urine has a very important diagnostic value in clinical events, so it is necessary to detect free phosphate ions in human urine. Scientific research results show that excessive magnesium ammonium phosphate, carbonate apatite, hydroxyapatite, calcium hydrogen phosphate dihydrate, tricalcium phosphate and other crystals in urine are one of the causes of urinary stones. We know that stone disease is accompanied by many pains such as renal colic, nausea and vomiting, hematuria, fever, etc., and even causes other diseases. Therefore, it is critical to maintain a certain concentration of phosphate in the urine. From this point of view, regular quantitative determination of phosphate in urine is essential. We can know the metabolic status of the organism and related pathological conditions by regularly measuring the content of phosphate radicals in the urine.

Regarding the determination of phosphate radicals in urine, the methods for detecting phosphate radicals in urine at home and abroad in the past can be summarized as the following:

1. Use ion chromatography to detect phosphate radicals in urine (L.Opliti, R.Chiaraluce, V.Consalvi, N.Cetulli and R.Scandurra, Clinica Chimica Acta, 184 (1989) 155-166; Journal of Chromatography, 433( 1988) 373-376;

2. Determination of phosphate in urine based on the strong inhibition of phosphate on calcium carbonate seeds (F. Grases and J.G. March, Analytica Chimica Acta, 229 (1990) 249-254);

3. Determination of phosphate in urine by sequential injection analysis using a single on-line calibration and photometry (D.G.Themelis, A.Economou, a.Tsiomlektsis and p.D.Tzanavaras, Analytical Biochemistry 330(2004) 193-198).

However, there are some problems with the above methods:

1. Many instruments are clinically used to measure phosphate in urine due to the narrow linear range, which requires a large degree of dilution of urine samples to measure.

2. Many methods of measuring phosphate in urine are measured indirectly by measuring some other components in urine. For example, the phosphate content can be determined by measuring the growth of calcium carbonate crystals in uroliths. This is both cumbersome and time-consuming, which brings a lot of inconvenience clinically.

3. Many methods for detecting phosphate in urine have low sensitivity, large deviation, or cannot give an accurate quantitative result.

Invention content:

The purpose of the present invention is to break through the previous traditional method for detecting phosphate in urine, and to invent a simple, sensitive, rapid and clinically applicable method for detecting phosphate in undiluted urine.

The invention provides a method for detecting phosphate in urine, which is based on ytterbium ions, and uses catechol violet as a color reagent to directly and quantitatively detect phosphate in urine in a HEPES buffer solution with a pH of 6.5-7.5. Phosphate, the specific detection steps are as follows:

1. Prepare a HEPES (10mM) buffer solution with a pH of 6.5-7.5, and prepare 2×10 ^-3 M Yb ³⁺ solution and 2×10 ^-3 M catechol violet solution;

2. Add 2ml of HEPES buffer solution to a clean UV cuvette, as a blank, use a micro-sampler to absorb 10-100μl of 2×10 ^-3 M catechol violet solution, and add it to this cuvette At this time, the solution turns from colorless to yellow, and it is detected on a UV-visible spectrophotometer, and there is a maximum absorption at 443nm;

3. In the above-mentioned cuvette, add the Yb ³⁺ solution whose molar number is 2 times that of the pyrocatechol violet solution. At this moment, the solution turns from yellow to blue, and detects on a UV-Vis spectrophotometer, and finds that the maximum absorption peak is formed by The above 443nm becomes 623nm;

4. Take a urine sample, gradually add it to the cuvette with a micro-injector, and detect it on the UV-Vis spectrophotometer while adding the sample. With the addition of the urine sample, the maximum absorption peak gradually changes from 623nm to 443nm The color of the solution also gradually changed from blue to yellow. When the absorption peak reached its maximum at 443nm and the color of the solution turned yellow completely, stop adding urine samples;

5. Calculate the phosphate radical content in urine according to [Yb ³⁺ ]×2ml×95/m _urine ×100%, where m _urine =V _urine .

The Yb ³⁺ solution is a YbCl ₃ solution or a Yb(ClO ₄ ) ₃ solution.

The Yb ³⁺ can be replaced by Nd ³⁺ , Sm ³⁺ , Gd ³⁺ , Er ³⁺ or Ho ³⁺ .

The present invention has the following advantages: 1, our detection object is the undiluted urine sample, saves the pretreatment procedure, brings very big convenience clinically; 2, detects on the ultraviolet-visible spectrophotometer, and the detection process is simple , and this effect can be clearly observed with the naked eye; 3. Although there are many other components in urine, our detection method shows good selectivity for phosphate, that is to say, the presence of other components does not interfere Our detection results; 4. Our detection is quantitative, has a large linear range, and the error is very small; obviously, this invention can be applied to clinically detect urine samples.

Description of drawings:

Figures 1 to 9 are UV-visible absorption diagrams for detecting phosphate radicals in urine corresponding to Examples 1 to 9.

Detailed ways:

Embodiment 1. prepare the HEPES (10mM) buffer solution of pH7.0, and prepare the YbCl ₃ solution of 2×10 ^-3 M and the pyrocatechol violet solution of 2×10 ^-3 M; Add the HEPES buffer solution of 2ml Put it into a clean ultraviolet cuvette, as a blank, draw 50 μl of 2×10 ^-3 M catechol violet solution with a micro-injector, add it to this cuvette, at this time, the solution turns from colorless to yellow, Detected on the HP8453 UV-Vis spectrophotometer, there is a maximum absorption at 443nm; in the above-mentioned cuvette, add 100 μl of 2×10 ^-3 M Yb ³⁺ solution, at this time, the solution turns from yellow to blue. It was detected on the instrument, and it was found that the maximum absorption peak changed from the above-mentioned 443nm to 623nm; adult urine samples were taken, gradually added to the cuvette with a micro-injector, and detected on the UV-Vis spectrophotometer while adding samples, as When the urine sample is added, the maximum absorption peak gradually changes from 623nm to 443nm, and the color of the solution gradually changes from blue to yellow. When the absorption peak reaches the maximum at 443nm and the color of the solution turns yellow completely, the amount of urine added It is 20 μl; calculation: 100 μl×2ml×95/20g×100%, the phosphate content in urine is 0.095%, and the ultraviolet-visible absorption diagram is shown in Figure 1.

Test 10 healthy adult urine samples according to Example 1, and the phosphate content in the urine is 0.0863-0.119%. If the 24-hour urine output of an adult is 1L, the range of inorganic phosphate excretion in adult urine is 0.863-1.19g, which is the same as the normal excretion of inorganic phosphate in urine (0.5-1.5g) in medical statistics. g/24 hours) are stable.

Embodiment 2. prepare the HEPES (10mM) buffer solution of pH6.5, all the other are the same as embodiment 1, the phosphate radical content is 0.095% in the urine, and the ultraviolet-visible absorption diagram is shown in Fig. 2.

Embodiment 3. Get one-year-old healthy child urine, all the other are the same as embodiment 1, calculate: 100 μ l * 2ml * 95/4g * 100%, the phosphate radical content is 0.38% in the urine, and the ultraviolet-visible absorption figure is shown in Fig. 3. From the above examples, we can see that the content of phosphate in children's urine is relatively high. This is because compared with adults, children have higher alkaline phosphatase activity and similar metabolic rate due to the fact that children are in the period of vigorous osteogenesis. Wait for the reason.

Embodiment 4. use the solution of Yb(ClO ₄ ) ₃ to get the urine of another adult, and all the other are the same as in Example 1, 100 μl × 2ml × 95/16g × 100%, so that the phosphate content in the urine is 0.119%. The UV-Vis absorption diagram is shown in Figure 4.

Embodiment 5. use the solution of _ErCl3 , all the other are the same as embodiment 1, 100 μ l * 2ml * 95/20g * 100%, the phosphate radical content is 0.095% in the urine, and the ultraviolet-visible absorption figure is shown in Fig. 5.

Example 6. Use the solution of NdClO ₄ ) ₃ , the rest are the same as in Example 1, 100 μl×2ml×95/20g×100%, the phosphate content in the urine is 0.095%, and the ultraviolet-visible absorption diagram is shown in Figure 6.

Example 7. Use the solution of SmClO ₄ ) ₃ , the rest are the same as in Example 1, 100 μl×2ml×95/20g×100%, the phosphate content in the urine is 0.095%, and the UV-visible absorption diagram is shown in Figure 7.

Embodiment 8. Use the solution of _GdCl3 , all the other are with embodiment 1, 100 μ l * 2ml * 95/16g * 100%, the phosphate radical content is 0.095% in the urine, and the ultraviolet-visible absorption diagram is shown in Figure 8.

Example 9. Use the solution of _HoCl3 , the rest are the same as in Example 1, 100 μl × 2ml × 95/16g × 100%, the phosphate content in the urine is 0.095%, and the UV-visible absorption diagram is shown in Figure 9.

Claims (3)

1, a kind of method that detects phosphate radical in the urine is characterized in that, this method comprises following detection step:

(1), preparation pH6.5-7.5 concentration is the HEPES buffer solution of 10mM, and prepares 2 * 10 ^-3The Yb of M ³⁺Solution and 2 * 10 ^-3The pyrocatechol violet solution of M;

(2), the HEPES buffer solution of 2ml is added in the clean ultraviolet cuvette, as blank, with microsyringe absorption 2 * 10 ^-3The pyrocatechol violet solution 10-100 μ l of M is added in this cuvette, and solution is detected on the UV, visible light spectrophotometer by colourless flavescence at this moment, and 443nm has absorption maximum;

(3), in above-mentioned cuvette, add 2 times of molal quantitys to the Yb of pyrocatechol violet solution ³⁺Solution, solution is detected on the UV, visible light spectrophotometer by xanthochromia indigo plant at this moment, finds that maximum absorption band becomes 623nm by above-mentioned 443nm;

(4), get urine sample, be added in this cuvette with microsyringe gradually, application of sample limit, limit is detected on the UV, visible light spectrophotometer, adding along with urine sample, maximum absorption band is become again to 443nm gradually by 623nm again, and the color of solution is also returned yellow by blue stain gradually, when absorption peak reaches maximum at 443nm, during the complete flavescence of the color of solution, stop to add urine sample;

(5), by [Yb ³⁺] * 2ml * 95/m _Urine* 100% calculates the content of phosphate radical in the urine, m in the formula _Urine=V _Urine

2, by the described a kind of method that detects phosphate radical in the urine of claim 1, it is characterized in that described Yb ³⁺Solution is YbCl ₃Solution or Yb (ClO ₄) ₃Solution.

3, by claim 1 or 2 described a kind of methods that detect phosphate radical in the urine, it is characterized in that described Yb ³⁺Can use Nd ³⁺, Sm ³⁺, Gd ³⁺, Er ³⁺Or Ho ³⁺Replace.

Images